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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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Global Ca2+ transport in NGF-differentiated PC12 cells is similar to that in undifferentiated PC12 cells. (A) Total Ca2+ transport with no Ca2+ transporters blocked is shown for undifferentiated (gray curve, Fig. 1) and NGF-differentiated (black curve, n = 48) PC12 control cells. (B) Ca2+ transport is shown for control (black curve, from A) and 4-blocked (gray curve, n = 62) NGF-differentiated PC12 cells. (C) Ca2+ transport is shown for NGF-differentiated PC12 cells treated with the 4-blocked protocol (light gray circles, from B) or with the 4-blocked protocol plus 10 μM BHQ (dark gray triangles, n = 16). The difference between these two curves (gray circles) might represent SPCA activity. (D) Ca2+ transport is shown for NGF-differentiated cells treated with the 4-blocked protocol (circles, from B) or pretreated with 50 μM GPN and then treated with the 4-blocked protocol (triangles, n = 18).
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fig6: Global Ca2+ transport in NGF-differentiated PC12 cells is similar to that in undifferentiated PC12 cells. (A) Total Ca2+ transport with no Ca2+ transporters blocked is shown for undifferentiated (gray curve, Fig. 1) and NGF-differentiated (black curve, n = 48) PC12 control cells. (B) Ca2+ transport is shown for control (black curve, from A) and 4-blocked (gray curve, n = 62) NGF-differentiated PC12 cells. (C) Ca2+ transport is shown for NGF-differentiated PC12 cells treated with the 4-blocked protocol (light gray circles, from B) or with the 4-blocked protocol plus 10 μM BHQ (dark gray triangles, n = 16). The difference between these two curves (gray circles) might represent SPCA activity. (D) Ca2+ transport is shown for NGF-differentiated cells treated with the 4-blocked protocol (circles, from B) or pretreated with 50 μM GPN and then treated with the 4-blocked protocol (triangles, n = 18).

Mentions: Fig. 6 A compares the Ca2+ transport curves for somata of NGF-differentiated (black line) and undifferentiated cells (gray line). They are very similar, with that for undifferentiated cells lying slightly above that for differentiated cells. When the 4-blocked protocol was applied to differentiated cells, we found that it abolished virtually all transport when [Ca2+]cyt was <1000 nM but left some residual Ca2+ transport above this value (Fig. 6 B). Adding 10 μM BHQ on top of the 4-blocked protocol (i.e., a 5-blocked experiment) decreased residual Ca2+ transport by ∼50% (Fig. 6 C). Thus, differentiation introduces a measurable component of Ca2+ clearance flux (out of the cytoplasm) that is not eliminated by TG but is BHQ sensitive. This flux could represent SPCA transporters on non-ER organelles of the secretory pathway. Unlike undifferentiated cells, the differentiated cells showed no depression of maximal Ca2+ rise after GPN treatment. GPN pretreatment had no effect on Ca2+ transport (Fig. 6 D), suggesting that the putative SPCA-containing compartment is not lysosomal.


Calcium transport mechanisms of PC12 cells.

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

Global Ca2+ transport in NGF-differentiated PC12 cells is similar to that in undifferentiated PC12 cells. (A) Total Ca2+ transport with no Ca2+ transporters blocked is shown for undifferentiated (gray curve, Fig. 1) and NGF-differentiated (black curve, n = 48) PC12 control cells. (B) Ca2+ transport is shown for control (black curve, from A) and 4-blocked (gray curve, n = 62) NGF-differentiated PC12 cells. (C) Ca2+ transport is shown for NGF-differentiated PC12 cells treated with the 4-blocked protocol (light gray circles, from B) or with the 4-blocked protocol plus 10 μM BHQ (dark gray triangles, n = 16). The difference between these two curves (gray circles) might represent SPCA activity. (D) Ca2+ transport is shown for NGF-differentiated cells treated with the 4-blocked protocol (circles, from B) or pretreated with 50 μM GPN and then treated with the 4-blocked protocol (triangles, n = 18).
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Related In: Results  -  Collection

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fig6: Global Ca2+ transport in NGF-differentiated PC12 cells is similar to that in undifferentiated PC12 cells. (A) Total Ca2+ transport with no Ca2+ transporters blocked is shown for undifferentiated (gray curve, Fig. 1) and NGF-differentiated (black curve, n = 48) PC12 control cells. (B) Ca2+ transport is shown for control (black curve, from A) and 4-blocked (gray curve, n = 62) NGF-differentiated PC12 cells. (C) Ca2+ transport is shown for NGF-differentiated PC12 cells treated with the 4-blocked protocol (light gray circles, from B) or with the 4-blocked protocol plus 10 μM BHQ (dark gray triangles, n = 16). The difference between these two curves (gray circles) might represent SPCA activity. (D) Ca2+ transport is shown for NGF-differentiated cells treated with the 4-blocked protocol (circles, from B) or pretreated with 50 μM GPN and then treated with the 4-blocked protocol (triangles, n = 18).
Mentions: Fig. 6 A compares the Ca2+ transport curves for somata of NGF-differentiated (black line) and undifferentiated cells (gray line). They are very similar, with that for undifferentiated cells lying slightly above that for differentiated cells. When the 4-blocked protocol was applied to differentiated cells, we found that it abolished virtually all transport when [Ca2+]cyt was <1000 nM but left some residual Ca2+ transport above this value (Fig. 6 B). Adding 10 μM BHQ on top of the 4-blocked protocol (i.e., a 5-blocked experiment) decreased residual Ca2+ transport by ∼50% (Fig. 6 C). Thus, differentiation introduces a measurable component of Ca2+ clearance flux (out of the cytoplasm) that is not eliminated by TG but is BHQ sensitive. This flux could represent SPCA transporters on non-ER organelles of the secretory pathway. Unlike undifferentiated cells, the differentiated cells showed no depression of maximal Ca2+ rise after GPN treatment. GPN pretreatment had no effect on Ca2+ transport (Fig. 6 D), suggesting that the putative SPCA-containing compartment is not lysosomal.

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