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Endocrinology Vol. 146, No. 11 4985-4993
Copyright © 2005 by The Endocrine Society

Dominant Role of Mitochondria in Calcium Homeostasis of Single Rat Pituitary Corticotropes

Andy K. Lee and Amy Tse

Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address all correspondence and requests for reprints to: Amy Tse, 9-70 Medical Sciences Building, Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. E-mail: amy.tse{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The rise in cytosolic free Ca2+ concentration ([Ca2+]i) is the major trigger for secretion of ACTH from pituitary corticotropes. To better understand the shaping of the Ca2+ signal in corticotropes, we investigated the mechanisms regulating the depolarization-triggered Ca2+ signal using patch-clamp techniques and indo-1 fluorometry. The rate of cytosolic Ca2+ clearance was unaffected by inhibitors of Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase (PMCA), slightly slowed by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, but dramatically slowed by mitochondrial uncouplers or inhibitor of mitochondrial uniporter. Measurements with rhod-2 revealed that depolarization-triggered increase in mitochondrial Ca2+ concentration. Thus, mitochondria have a dominant role in cytosolic Ca2+ clearance. Using the Mn2+ quench technique, we found the presence of a continuous basal Ca2+ influx in corticotropes. This basal Ca2+ influx was balanced by the combined actions of mitochondrial uniporter and PMCA and SERCA pumps. Inhibition of the mitochondrial uniporter or PMCA or SERCA pumps elevated basal [Ca2+]i. Using membrane capacitance measurement, we found that the change in the shape of the depolarization-triggered Ca2+ signal after mitochondrial inhibition was associated with enhancement of the exocytotic response. Thus, mitochondria have a dominant role in the regulation of Ca2+ signal and exocytosis in corticotropes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CORTICOTROPE IN THE anterior pituitary gland is a key component in the hypothalamic-pituitary-adrenal axis that mediates the endocrine response to stress. The cytosolic Ca2+ signal controls diverse cellular functions in corticotropes. In particular, intracellular Ca2+ concentration ([Ca2+]i) regulates the exocytosis of ACTH-containing secretory granules (1) and affects the expression of the proopiomelanocortin gene (2). The ACTH secretagogue, CRH causes a sustained (minutes) elevation of [Ca2+]i in corticotropes (3, 4, 5, 6) via a cAMP-dependent inhibition of background K+ channels, which, in turn, leads to depolarization and activation of voltage-gated Ca2+ channels (3, 4, 5). Another ACTH secretagogue, arginine vasopressin (AVP) triggers Ca2+ release from the inositol trisphosphate (IP3)-sensitive Ca2+ stores and evokes a transient and plateau pattern of Ca2+ signal (7). Interestingly, stimulation of {alpha}-adrenergic receptors, which also activates Ca2+ release from IP3-sensitive Ca2+ stores, triggers slow [Ca2+]i oscillation in corticotropes (8). The ability of various agonists to elicit different patterns of Ca2+ signal suggests that Ca2+ homeostasis in corticotropes must involve a repertoire of mechanisms.

In most cells, several major mechanisms are available for transporting Ca2+ (9). These include Na+/Ca2+ exchanger (NCX), the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump, plasma membrane Ca2+-ATPase (PMCA) pump, and the mitochondria. Different cell types appear to use various combinations of these Ca2+-transport mechanisms to regulate their Ca2+ signals. For example, in sympathetic neurons as well as adrenal chromaffin cells, mitochondria rapidly uptake Ca2+ during voltage-gated Ca2+ entry to limit both the amplitude and duration of the Ca2+ signal (10, 11). On the other hand, in endocrine cells such as the mouse pancreatic ß-cells, the mitochondria contribute little to the Ca2+ dynamics; instead, the SERCA pump is the dominant cytosolic Ca2+ clearance mechanism (12). The shape of the Ca2+ signal in mouse pancreatic ß-cell is strongly regulated by the SERCA pump-mediated Ca2+ uptake and subsequent release of Ca2+ from the endoplasmic reticulum (12, 13). In pituitary gonadotropes, both the SERCA pump and mitochondria are involved in the clearance of Ca2+ from the cytosol (14, 15, 16). The contribution of the various mechanisms in regulating Ca2+ homeostasis in primary corticotropes is not known. A study of the clonal pituitary cell line (AtT-20) of mouse corticotropes has suggested that the PMCA pump may be the primary cytosolic Ca2+ clearance mechanism and that neither SERCA pump nor mitochondrial Ca2+ uptake contributes significantly to cytosolic Ca2+ clearance (17). An interesting aspect of corticotropes is that these cells may be adapted to stimulus-secretion coupling of long duration. The plasma level of ACTH is pulsatile (18). In rats, during the diurnal ACTH surge, elevations of the plasma ACTH can persist for 1 h (19, 20). The ability of corticotropes to secrete over a long period of time is also shown by the observation that the CRH-evoked ACTH release can be maintained for hours (21). Because ACTH secretion from corticotropes is dependent on [Ca2+]i rise (1, 21), it is conceivable that in these cells, [Ca2+]i rise is maintained during the prolonged ACTH secretion. Thus, corticotropes may have to adopt specific Ca2+-transport mechanisms to prevent Ca2+ overload. In this study, we investigated the contributions of the NCX, SERCA, PMCA and mitochondria in the Ca2+ dynamic of corticotropes. We found that the mitochondria are the dominant mechanism for cytosolic Ca2+ clearance in corticotropes. Inhibition of mitochondrial Ca2+ uptake dramatically modifies the depolarization-triggered Ca2+ transient as well as the exocytotic response of corticotropes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparation
The anterior lobe of the pituitary glands were removed from male Sprague-Dawley rats (age 5–6 wk) killed with an overdose of halothane in accordance with standards of the Canadian Council on Animal Care. Anterior pituitary glands were dissociated enzymatically using collagenase and trypsin as previously described (22). Single corticotropes were identified from the heterogeneous pituitary cell population by reverse hemolytic plaque assay (23). The procedures were similar to that described previously (8). Briefly, dissociated pituitary cells were suspended in DMEM (Life Technologies, Inc., Grand Island, NY) that contained 0.1% (wt/vol) BSA (Sigma, St. Louis, MO). The pituitary cell suspension was then mixed with one-third the volume of 12% (vol/vol) sheep erythrocytes (Colorado Serum Co., Denver, CO) in 0.9% (wt/vol) NaCl. The erythrocytes were previously conjugated with Staphylococcus aureus-derived protein A (Sigma), using 0.2 mg ml–1 CrCl3 as a catalyst. The cell mixture was incubated with a mixture of 10 nM CRH and 100 nM AVP and rabbit polyclonal antibodies to rat ACTH (1:20 dilution; gift from Dr. R. J. Kemppainen, Auburn University, Auburn, AL) for 3 h at 37 C. Plaques were formed by a 30-min exposure to guinea pig complement at 1:50 dilution. The cells were maintained under standard culture condition in a DMEM supplemented with 10% (vol/vol) horse serum, 50 U ml–1 penicillin G, and 50 mg ml–1 streptomycin. Recordings were made in cells cultured for 2–4 d after plaque formation.

Chemicals and solutions
The standard bath solution contained (in mM): 150 NaCl, 10 Na-HEPES, 8 glucose, 2.5 KCl, 2 or 5 CaCl2, and 1 MgCl2 (pH 7.4). In experiments involving inhibition of PMCA pump, the pH of the standard bath solution was raised to 8.8. For experiments with Ca2+-free bath solution, CaCl2 was omitted from the standard bath solution and MgCl2 was increased to 3 mM. For Na+-free solution, NaCl in the standard solution was replaced with N-methyl-glucamine-Cl. The standard pipette solution contained (in mM): 120 K-aspartate, 20 K-HEPES, 20 KCl, 1 MgCl2, 2 Na2ATP, 0.1 Na4GTP (pH 7.4). For perforated patch recording, nystatin (250 µg/ml) was included in the pipette solution. In experiments involving high concentrations of ATP, Na2ATP was increased to 10 mM and MgCl2 to 5 or 9 mM. For membrane capacitance measurement, the pipette solution contained (in mM): 135 Cs-aspartate, 20 tetraethylammonium-Cl, 20 Cs-HEPES, 2.5 MgCl2, 5 Na2-ATP, 0.1 Na4GTP (pH 7.4). Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were obtained from Calbiochem (San Diego, CA). Indo-1 K+ salt and indo-1 acetoxymethyl ester (AM) were obtained from Teflabs (Austin, TX). Rhod-2 AM was obtained from Molecular Probes (Eugene, OR). All other chemicals were obtained from Sigma (Oakville, Ontario, Canada).

Electrophysiology
Single corticotropes were recorded with the whole-cell or perforated patch clamp configuration with an EPC-9 patch clamp amplifier. Cells were voltage clamped at –70 mV and voltage pulses (500 msec) to 10 mV were applied to activate voltage-gated Ca2+ channels and elicit Ca2+ transients. Holding potential and voltage pulses were controlled, and data were acquired using the EPC-9 Pulse software (HEKA Electronics, Mahone Bay, Nova Scotia, Canada) on a Windows-based computer. Membrane-capacitance measurement was made using the built-in lock-in software in Pulse. A 1-kHz, 15-mV peak-to-peak sinusoid and the sine wave plus direct current method were used to calculate the membrane capacitance. The pipettes were made from hematocrit glass (VWR Scientific Canada Ltd., London, Ontario, Canada) and the resistance was 2–5 M{Omega} after filling with pipette solution. All experiments were performed at room temperature (20–23 C). A –10 mV junction potential was corrected throughout.

Measurement of [Ca2+]i
Except for the Mn2+ quench experiments, [Ca2+]i measurement was made with the Ca2+-sensitive dye, indo-1 (0.1 mM), which was included in the pipette solution and dialyzed into the cell. Details of the instrumentation and procedures of [Ca2+]i measurement were as described previously (3). Briefly, the dye was excited by 365-nm light from a HBO 100-W mercury lamp via a x40, 1.3 NA UV fluor oil objective lens (Nikon). Emission fluorescence at 405 ± 35 nm and 495 ± 45 nm was collected with two photomultiplier tubes (Hamamatsu H3460-04). The output of the photomultiplier tubes were converted to transistor transistor logic (TTL) pulses and counted by a CYCTM-10 counter card (Cyber Research Inc., Branford CT) installed in a Windows-compatible computer. [Ca2+]i was calculated from the ratio (R) of the fluorescence at 405 and 495 nm according to the equation: [Ca2+] = K* x (R – Rmin)/(Rmax – R), where Rmin is the ratio when Ca2+ is strongly chelated with EGTA, Rmax is the ratio when 15 mM free Ca2+ is present, and K* is a constant determined empirically (24). Rmin was measured in cells loaded with (in mM): 52 KAsp, 50 K-HEPES, 50 EGTA, and 10 KCl (pH 7.4). Rmax was measured in cells loaded with (in mM): 136 K-asparate, 50 K-HEPES, and 15 CaCl2 (pH 7.4). K* was calculated from the above equation using ratio values obtained cells loaded with (in mM): 60 K-asparate, 50 K-HEPES, 20 EGTA, 15 CaCl2 (pH 7.4), which had a calculated free [Ca2+]i of 212 nM at 24 C (25).

Measurement of extracellular Ca2+ influx using Mn2+ quenching
Corticotropes were incubated with 5 µM indo-1 AM in standard bath solution for 20 min and then in dye-free solution for 5 min at 37 C. Cells were then recorded with the perforated patch-clamp configuration. Extracellular Mn2+ enters into the cell via Ca2+-permeable pathways and causes quenching (reduction) in indo-1 fluorescence. Because the fluorescence at the relatively broad band around 405 nm (F405) is not sensitive to changes in [Ca2+]i [near the isosbestic wavelength of indo-1 (26)], the rate of decrease in F405 by Mn2+ reflects the rate of extracellular Ca2+ influx (27, 28). On the other hand, [Ca2+]i is determined by the ratio of F405/F495 (as described above). Rise in [Ca2+]i results in decrease in F495. Mn2+ quenches both F405 and F495 to the same extent and thus has no effect on the ratio of F405/F495 and [Ca2+]i. The rate of Mn2+ quenching of the fluorescence was calculated from the slope of a linear fit to individual segments of F405. The slope was then normalized to the cell surface area (estimated from the cell membrane capacitance) of individual corticotropes.

Measurement of mitochondrial Ca2+ signal using rhod-2 fluorescence
Corticotropes were incubated with 2 µM rhod-2 AM for 45–60 min at room temperature. At the end of the incubation, punctate fluorescent staining within the cell was observed, suggesting the dye had localized to the mitochondria. Cells were then incubated with dye-free solution and recorded with the whole-cell patch-clamp configuration. This allowed dialysis of the cytosolic rhod-2 out of the cell via the patch pipette, thus reducing contamination to the mitochondrial signal. Rhod-2 was excited at 535 nm, and fluorescence was monitored at wavelengths greater than 590 nm using a long-pass filter.

Calculations and statistics
Functions in the Microcal Origin program 6.0 (Microcal Software) were employed for all statistical and curve-fitting procedures. The time constant of Ca2+ clearance was estimated by fitting the decay of the Ca2+ signal with a single exponent. The rate of Mn2+ quench was calculated from the slopes of the linear fit to individual segments of the fluorescence at 405 nm. A Student’s t test was used in comparisons of mean values between two populations of cells. Any difference with P < 0.05 was considered statistically significant. All values shown were means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course of cytosolic Ca2+ clearance
In the current study, we defined basal [Ca2+]i as the steady-state [Ca2+]i level (maintained for >10 sec) before the delivery of depolarization to trigger a Ca2+ transient, and the rate of cytosolic Ca2+ clearance was estimated from the time course of the decay of the depolarization-triggered Ca2+ transient. Because the activities of different mechanisms of cytosolic Ca2+ clearance may be affected by basal [Ca2+]i, we first examined whether the level of basal [Ca2+]i could affect the rate of cytosolic Ca2+ clearance. In this experiment, we compared the time course of the decay of the depolarization-triggered Ca2+ transient in the same cell when basal [Ca2+]i was elevated to two different levels. A representative example of this experiment is shown in Fig. 1Go. The cell was voltage clamped at –70 mV, and a depolarizing voltage step was applied to trigger a Ca2+ transient. After the return of the Ca2+ signal to the resting level, the holding potential was changed to –40 mV. This resulted in a small activation of the voltage-gated Ca2+ channel, and the basal [Ca2+]i rose by approximately 0.15 µM. Another depolarizing voltage step was then applied to trigger a second Ca2+ transient. Note that the decay phase of each depolarization-triggered Ca2+ transient could be well described with a single exponential function. Therefore, we estimated the rate of Ca2+ clearance from the time constant of the single exponential function fitted to the decay phase of the Ca2+ transient. In this cell, the time constant of the first Ca2+ transient was 4.7 sec. After the elevation in basal [Ca2+]i, the time constant was reduced to 3.8 sec. In 10 cells examined, changing the holding potential from –70 to –40 mV raised the basal [Ca2+]i from 0.21 ± 0.03 to 0.39 ± 0.04 µM (P < 0.05 for Student’s t test). In these cells, the initial time constant was 5.9 ± 0.5 sec, and after elevation of basal [Ca2+]i, the time constant was 5.3 ± 0.4 sec (n = 10; P < 0.05 for Student’s t test). This result suggests that there was a small decrease in the time constant of Ca2+ decay after a modest elevation in basal [Ca2+]i. We also compared time constants of Ca2+ clearance for cells with different resting [Ca2+]i. For cells with resting [Ca2+]i of less than 0.2 µM, the mean time constant of Ca2+ clearance was 5.3 ± 0.2 sec (n = 139), whereas for cells with basal [Ca2+]i between 0.4 and 0.6 µM, the mean time constant of Ca2+ clearance was 4.9 ± 0.3 sec (n = 26). Although the general trend of reduction in the time constant of Ca2+ decay with elevation in basal [Ca2+]i was observed in these two groups of cells, there was no significant difference in their time constants (P > 0.05 for Student’s t test). Therefore, we conclude that in corticotropes, when the basal [Ca2+]i was elevated to 0.6 µM, the time constant of Ca2+ clearance was slightly reduced or essentially unaffected.



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  		FIG. 1. Moderate rise in basal [Ca2+]i did not slow Ca2+ clearance. A corticotrope was held –70 mV and stimulated with a single voltage step to 10 mV. When the holding potential of the cell was changed to –40 mV to activate voltage-gated Ca2+ channels, there was a rise in basal [Ca2+]i. A second voltage step was then applied. The time course of the two Ca2+ transients could be fitted with a single exponential function (thick line). The time constant of decay for the first and second Ca2+ transient was 4.7 and 3.8 sec, respectively. Note that for all the figures shown in this study, the duration of a single voltage step was 500 msec.

 
Influence of NCX and SERCA pump on Ca2+ signal
To investigate the role of NCX in regulating Ca2+ homeostasis in corticotropes, we replaced Na+ in the extracellular solution by an impermeant ion N-methyl-glucamine. As shown in Fig. 2AGo, the absence of extracellular Na+ did not have any major effect on the basal [Ca2+]i or the rate of the decay of the depolarization-triggered Ca2+ transient in corticotropes. In 16 cells examined, the removal of extracellular Na+ caused a small increase in the basal [Ca2+]i (43 ± 9 nM), and the rate of Ca2+ clearance after Na+ removal was unchanged (1.00 ± 0.05-fold of the control; P > 0.05 for Student’s t test).



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  		FIG. 2. Influence of NCX and SERCA pump on [Ca2+]i. A, The removal of extracellular Na+ did not affect the basal [Ca2+]i nor the decay of the Ca2+ transient. Each of the Ca2+ transients was stimulated by five voltage steps. B, Inhibition of SERCA pump by BHQ (20 µM) caused a small elevation of [Ca2+]i and a slight slowing of the decay of the Ca2+ transient. Note that BHQ reduced the amplitude of the depolarization-triggered transient. Therefore, a second depolarization (three voltage steps) was applied in BHQ to elicit a larger Ca2+ transient for estimation of Ca2+ clearance. Except for the second Ca2+ transient in BHQ, all other Ca2+ transients were evoked with a single voltage step.

 
Rat corticotropes have IP3-sensitive stores that can be released by secretagogues such as AVP and norepinephrine (7, 8). Because SERCA pump is responsible for Ca2+ reuptake into the intracellular stores, we investigated the role of the SERCA pumps with a reversible SERCA pump inhibitor, BHQ. The EC50 of BHQ for inhibition of SERCA pump was reported to be 1–5 µM (29, 30, 31). As shown in Fig. 2BGo, application of BHQ (20 µM) resulted in a small elevation of the basal [Ca2+]i and some slowing in the decay of the depolarization-triggered Ca2+ transient. Note that in the presence of BHQ, the amplitude of the depolarization-triggered Ca2+ transient was smaller. However, this effect of BHQ was not related to SERCA-pump inhibition but probably due to an inhibitory action of BHQ on voltage-gated Ca2+ channels, because BHQ at 10–60 µM have been reported to inhibit Ca2+ channels in other cell types (32, 33, 34). Therefore, more voltage steps were applied in BHQ to evoke a Ca2+ transient with amplitude more comparable to the control (Fig. 2BGo). In eight cells examined, BHQ raised the basal [Ca2+]i by 0.16 ± 0.02 µM and caused a significant slowing in the rate of Ca2+ clearance rate (by 1.21 ± 0.05-fold; P < 0.05 for Student’s t test).

Influence of the mitochondria on Ca2+ signal
In contrast to the NCX and SERCA pump, we found that mitochondria dramatically affected Ca2+ homeostasis in rat corticotropes. We first employed the mitochondrial uncoupler, cyanide which inhibits the activity of complex IV in the respiratory chain, thereby preventing the recharging of the mitochondrial membrane potential. As shown in Fig. 3AGo, cyanide caused an elevation in the basal [Ca2+]i and dramatically slowed the decay of the depolarization-triggered Ca2+ transient. This action of cyanide is reversible. After the removal of cyanide, both the basal [Ca2+]i and the rate of decay of the depolarization-triggered Ca2+ transient returned to the control level (Fig. 3AGo). In 16 cells examined, the mean rise in basal [Ca2+]i by cyanide was 0.75 ± 0.15 µM (ranging from 60 nM to >1 µM). Because the rate of cytosolic Ca2+ clearance was essentially unaffected by basal [Ca2+]i up to 0.6 µM (Fig. 1Go), we restricted the analysis of Ca2+ clearance to cells with basal [Ca2+]i < 0.6 µM (in the presence of cyanide). In five cells examined, cyanide slowed the rate of Ca2+ clearance by 3.6 ± 0.8-fold (P < 0.05 for Student’s t test). As shown in Fig. 3BGo, the metabolic inhibitor CCCP that directly collapses the mitochondrial membrane potential, also raised basal [Ca2+]i and slowed Ca2+ clearance. In 17 cells examined, CCCP raised the basal [Ca2+]i by 1.46 ± 0.32 µM. In the seven cells where the basal [Ca2+]i in the presence of CCCP was less than 0. 6 µM, CCCP slowed the rate of Ca2+ clearance by 2.7 ± 0.5-fold (P < 0.05 for Student’s t test). Because the effects of CCCP were generally less reversible than cyanide, we employed cyanide in the majority of the following experiments.



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  		FIG. 3. Mitochondrial uncouplers raised basal [Ca2+]i and slowed Ca2+ clearance. A, Action of cyanide (5 mM). All Ca2+ transients were elicited by a single voltage step. B, Action of CCCP (2 µM). Two voltage steps were applied before and after the removal of CCCP. To match the amplitude of the Ca2+ transient before CCCP, a single voltage step was applied in the presence of CCCP.

 
The large effects of mitochondrial uncouplers on the basal [Ca2+]i and Ca2+ clearance in rat corticotropes suggest that Ca2+ uptake into the mitochondria is particularly important for Ca2+ homeostasis in these cells. To examine whether Ca2+ was taken into the mitochondria after depolarization, we measured changes in mitochondrial Ca2+ signal with rhod-2 fluorescence. The membrane-permeant form (AM) of rhod-2 has a net-positive charge and preferentially accumulates in the mitochondria, where it is cleaved and made membrane impermeant by endogenous esterases. Because rhod-2 is sensitive to changes in Ca2+, a change in rhod-2 fluorescence reflects a change in mitochondria Ca2+ concentration. As shown in Fig. 4AGo, depolarization triggered an increase in rhod-2 fluorescence and this increase was reversibly reduced by cyanide. Similar results were observed in five cells. These results suggest that during voltage-gated Ca2+ entry, some Ca2+ was rapidly taken up into the mitochondria. Mitochondrial Ca2+ uptake is mediated via a uniporter, which is sensitive to ruthenium red (10). Therefore, we asked whether the uptake of Ca2+ into the mitochondria after depolarization was mediated by the uniporter. As shown in Fig. 4BGo, the depolarization-triggered increase in rhod-2 fluorescence was reduced by the presence of ruthenium red (50 µM) in the whole-cell pipette. After approximately 6 min of dialysis, the amplitude of the increase in rhod-2 fluorescence was reduced to 25 ± 2% (n = 3). In contrast, for the time-matched controls (no ruthenium red in the whole-cell pipette; e.g. Fig. 4CGo), the amplitude of the increase in rhod-2 fluorescence was 77 ± 18% (n = 3), significantly larger than the cells recorded with ruthenium red. To examine whether the slowing of cytosolic Ca2+ clearance by mitochondrial uncoupler was related to the inhibition of mitochondrial Ca2+ uptake, we asked whether inhibition of the mitochondrial uniporter could slow down cytosolic Ca2+ clearance. As shown in Fig. 4DGo, the decay of the depolarization-triggered Ca2+ transient gradually slowed down as ruthenium red (10 µM) was dialyzed into the cell. In contrast, there was less slowing of the decay of the depolarization-triggered Ca2+ transient in the time-matched control shown in Fig. 4EGo, even though the same numbers of depolarizations were applied. In 13 cells examined with ruthenium red (10 µM) in the pipette solution, the time constant of the decay of the depolarization-triggered Ca2+ transient at approximately 4 min after the establishment of whole-cell configuration was 12.8 ± 1.2 sec. In contrast, for cells recorded without ruthenium red, the time constant of the decay of the depolarization-triggered Ca2+ transient at approximately 4 min after the establishment of whole-cell configuration and after the same numbers of depolarization as the cells recorded with ruthenium red was 4.8 ± 0.5 sec (n = 8), approximately 2.6-fold faster than the cells recorded with ruthenium red (P < 0.05 for Student’s t test). As shown in Fig. 4DGo, the dialysis of ruthenium red into the cell also resulted in a gradual increase in basal [Ca2+]i. In cells dialyzed with ruthenium red (10 µM) for 4 or 7 min, the basal [Ca2+]i increased by 0.13 ± 0.3 µM (n = 13) and 0.22 ± 0.03 µM (n = 15), respectively. For control cells, the basal [Ca2+]i increased by 0.10 ± 0.4 µM (n = 8) and 0.12 ± 0.04 µM (n = 10), respectively, after 4 or 7 min of whole cell. The increase in basal [Ca2+]i in the cells with ruthenium red was significantly higher than the increase in time-matched control cells only after 7 min of whole-cell dialysis (P < 0.05 for Student’s t test) and the magnitude of the increase in basal [Ca2+]i was also smaller than those elicited by cyanide or CCCP (Fig. 3Go). Consistent with this, application of cyanide after approximately 7 min of dialysis of ruthenium red (10 µM) into the cell caused a further increase in basal [Ca2+]i as well as slowing of the cytosolic Ca2+ clearance. In the presence of ruthenium red, cyanide further elevated the basal [Ca2+]i by 0.46 ± 0.13 µM (n = 10) and slowed the cytosolic Ca2+ clearance by 1.8 ± 0.1-fold (n = 4; P = 0.05 for Student’s t test). We observed similar results even after we raised ruthenium red to 50 µM (n = 5). Note that, as shown in Fig. 4BGo, the inclusion of 50 µM ruthenium red in the whole-cell pipette could largely reduce the depolarization-triggered increase in rhod-2 signal within approximately 4 min of whole-cell dialysis. Thus, it is unlikely that the larger effect of cyanide is due to the insufficient block of uniporter activity by ruthenium red. Instead, it may be related to secondary effects of metabolic inhibition by cyanide. One effect associated with metabolic inhibition is ATP depletion, because the poisoned mitochondria can no longer produce ATP. In our experiments, cells were supplied with ATP (2 mM) via the whole-cell pipette. Nevertheless, it is possible that this concentration of ATP was insufficient to fully compensate the loss of the ATP production in the mitochondria. A reduction in cellular ATP level would lead to a decrease in the activities of Ca2+-ATPases and, thus, affect both the SERCA and PMCA pumps. Therefore, we examined whether the effect of cyanide could be reduced when the concentration of ATP in the whole-cell pipette was increased to 10 mM. Under this condition, the cyanide-mediated increase in basal [Ca2+]i was 0.28 ± 0.05 µM (n = 20), significantly (P < 0.05 for Student’s t test) smaller than those recorded with 2 mM ATP (0.75 ± 0.15 µM; n = 16). The slowing in the rate of cytosolic Ca2+ clearance by cyanide in cells recorded with 10 mM ATP was 2.7 ± 0.6-fold (n = 9), smaller but not significantly (P > 0.05 for Student’s t test) different from the slowing observed in cells with 2 mM ATP (3.6 ± 0.8-fold; n = 5). Thus, raising the concentration of ATP to 10 mM could attenuate the large effect of cyanide on basal [Ca2+]i observed with 2 mM ATP in the pipette solution. However, the slowing of the decay of the depolarization-triggered Ca2+ transient by cyanide was less sensitive to the concentration of ATP in the pipette.



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  		FIG. 4. Uptake of Ca2+ via mitochondrial uniporter. A, Depolarization-triggered increase in mitochondrial Ca2+ signal. Cyanide reversibly inhibited the depolarization-mediated increase in mitochondrial Ca2+ signal (monitored by rhod-2 fluorescence; a.u., arbitrary units). The depolarization (indicated by the arrow) comprised five voltage steps. B, Inhibition of mitochondrial uniporter by ruthenum red reduced the depolarization-mediated increase in mitochondrial Ca2+ signal. Ruthenium red (50 µM) was dialyzed into cell via the whole-cell pipette. The depolarization (comprised three voltage steps) was delivered at approximately 2, 4, and 6 min (indicated by the arrows) after whole cell. C, Depolarization-triggered increase in mitochondrial Ca2+ signal in a time-matched control cell. The three depolarizations were delivered at about the same time after whole cell as in B. D, Inhibition of mitochondrial uniporter resulted in rise in basal [Ca2+]i and slowing of Ca2+ clearance. Ruthenium red (10 µM) was dialyzed into cell via the whole-cell pipette, and the depolarization (a single voltage step) was delivered at approximately 1, 2, 3, and 4 min after whole cell (denoted by arrows). E, In time-matched control cell, there was a small slowing of Ca2+ clearance.

 
Mechanisms involved in the regulation of basal [Ca2+]i
Because the mitochondrion itself is a Ca2+ store, the disruption of mitochondrial functions by cyanide can lead to mitochondrial Ca2+ release and, thus, a rise in basal [Ca2+]i. To consider this possibility, we investigated whether the cyanide-mediated rise in basal [Ca2+]i was associated with intracellular Ca2+ release. Surprisingly, Fig. 5AGo shows that the cyanide-stimulated rise in basal [Ca2+]i was strongly dependent on the presence of extracellular Ca2+. In the absence of extracellular Ca2+, cyanide elicited a very small rise in basal [Ca2+]i. When extracellular Ca2+ was restored, application of cyanide to the same cell evoked a robust rise in basal [Ca2+]i. In eight cells examined, the cyanide-mediated [Ca2+]i rise in the presence of extracellular Ca2+ was 1.04 ± 0.28 µM, significantly (P < 0.05 for Student’s t test) larger than that elicited in the absence of extracellular Ca2+ (0.15 ± 0.07 µM; n = 8). Evidently, the cyanide-mediated rise in basal [Ca2+]i requires extracellular Ca2+ influx. We then asked whether the cyanide-mediated rise in basal [Ca2+]i was associated with any stimulation of extracellular Ca2+ influx via opening of ion channels at the plasma membrane. For this experiment, we blocked the K+ channels by including Cs+ and tetraethylammonium+ in the pipette solution. In addition, apamin (0.5 µM) was included in the extracellular solution to inhibit the small conductance Ca2+-activated K+ channels in corticotropes (7). Figure 5BGo shows a simultaneous measurement of [Ca2+]i and holding current when the cell was voltage-clamped at –70 mV. In this example, application of cyanide evoked a large rise in [Ca2+]i. Note, however, that the rise in basal [Ca2+]i was not associated with any major change in holding current (< 5 pA). Similar results were observed in 15 other cells. For all the experiments described above, cells were voltage clamped at –70 mV and, as shown in our previous study (3), no voltage-gated Ca2+ channel was activated at this potential. Nevertheless, it is possible that cyanide may affect the activation of some low-threshold voltage-gated Ca2+ channel at this potential, thus affecting the basal [Ca2+]i. Therefore, we examined whether the cyanide-mediated rise in basal [Ca2+]i could be reduced by voltage clamping the cell at more negative potential. In this experiment, the effect of cyanide was compared on the same cells when the holding potential was changed from –70 or –90 mV. In seven cells examined, the cyanide-mediated [Ca2+]i rise increased by 0.16 ± 0.04 µM when the holding potential was changed from –70 to –90 mV. Thus, it is unlikely that the cyanide-mediated [Ca2+]i involves any activation of voltage-gated Ca2+ channels.



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  		FIG. 5. The cyanide-mediated rise in basal [Ca2+]i required extracelluar Ca2+ influx. A, In the absence of extracellular Ca2+, cyanide triggered only a very small rise in [Ca2+]i. After the restoration of extracellular Ca2+, cyanide stimulated a much larger increase in basal [Ca2+]i. Note that in the absence of extracellular Ca2+, depolarization did not cause any change in [Ca2+]i. B, The cyanide-mediated rise in basal [Ca2+]i was not accompanied by any detectable change in membrane current. The corticotrope was held at –70 mV. Only the holding current at –70 mV was shown.

 
The results above suggest that the cyanide-mediated rise in basal [Ca2+]i was not associated with large activation of currents. One possibility is that the cyanide-mediated [Ca2+]i rise involves Ca2+ fluxes (e.g. via ion exchanger) with no net-charge transfer. Alternatively, some Ca2+-permeable channels, such as the capacitative Ca2+ channels, are very small and may be difficult to measure under our experimental conditions. Consistent with the notion of capacitative Ca2+ entry, the cyanide-mediated [Ca2+]i rise increased when the membrane potential was more negative (thus increasing the driving force for extracellular Ca2+ entry). Therefore, we used the Mn2+ quench technique to further examine whether the cyanide-mediated [Ca2+]i rise was associated with the activation of extracellular Ca2+-influx pathways. We loaded corticotropes with indo-1 AM and then recorded the cells with the perforated-patch configuration. Because the indo-1 fluorescence at 405 nm (F405) is near the isobestic point of the indo-1 spectrum (26), F405 is not sensitive to changes in [Ca2+]i. As shown in Fig. 6AGo, a depolarization-triggered Ca2+ entry did not affect F405 but caused a transient reduction in F495. Because Mn2+ binds to indo-1 with high affinity and quenches indo-1 fluorescence, its entry into a cell would manifest as a decrease in indo-1 fluorescence. Furthermore, Mn2+ enters the cell through Ca2+-permeable pathways; therefore, an acceleration in the rate of indo-1 fluorescence decrease (quench rate) reflects the activation of Ca2+-influx pathway. Application of Mn2+ (0.5 mM) resulted in a slow decrease in F405, reflecting a basal leakage of Mn2+ into the cell, but it was not accompanied by any rise in [Ca2+]i. (Fig. 6AGo). Application of cyanide caused a rise in basal [Ca2+]i, but it was not accompanied by any acceleration in the rate of decrease of F405. The results from six cells are summarized in Fig. 6BGo. In these cells, cyanide raised basal [Ca2+]i by 0.60 ± 0.12 µM, but the rate of decrease of F405 was 0.18 ± 0.03 arbitrary units sec–1 pF–1 before cyanide, not significantly (P > 0.05) different from the rate recorded in the presence of cyanide (0.19 ± 0.05 arbitrary units sec–1 pF–1; n = 6). Clearly, cyanide did not elicit any additional Mn2+ influx. This observation suggests that at basal condition, there is a significant and continuous extracellular Ca2+ influx into corticotropes. Consistent with this, the removal of extracellular Ca2+ typically resulted in a lowering of basal [Ca2+]i in corticotropes. In eight cells examined, the basal [Ca2+]i was reduced by 0.13 ± 0.03 µM upon removal of extracellular Ca2+. Under control conditions, this basal Ca2+ entry is effectively buffered and removed, such that the basal [Ca2+]i in the cell is maintained at a low level. In the presence of cyanide, the inhibition of mitochondrial Ca2+ uptake reduced the buffering of Ca2+ and contributed to the rise in basal [Ca2+]i. In addition, the inhibitory action of cyanide on metabolism may lead to a decrease in ATP production, which, in turn, can reduce the activities of SERCA and PMCA pumps. As shown in Fig. 2BGo, the inhibition of SERCA pumps resulted in a rise in basal [Ca2+]i. To examine whether PMCA pumps also contribute to the regulation of basal [Ca2+]i, we reduced PMCA activity by exposing the cells to an extracellular solution with pH 8.8. This experimental approach is based on the Ca2+/H+ exchanger property of the PMCA pump. By lowering the H+ concentration in the extracellular solution (raising the pH to 8.8), the activities of the PMCA pump can be reduced (35). Such a manipulation has been used successfully to reduce PMCA pump activity in mouse pancreatic ß-cells (12). When corticotropes were exposed to a pH 8.8 extracellular solution, there was a rise in basal [Ca2+]i (0.18 ± 0.04 µM; n = 14), but the rate of Ca2+ clearance was not significantly affected (1.1 ± 0.1-fold of the control; n = 12).



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  		FIG. 6. The cyanide-mediated rise in basal [Ca2+]i was not due to the activation of additional extracellular Ca2+ influx. A, A corticotrope was voltage-clamped at –70 mV in the perforated patch configuration and a single voltage step was applied at the time indicated by arrows. The cell was loaded with indo-1 AM. Indo-1 fluorescence was monitored at 405 nm and 495 nm (a.u., arbitrary units). Note that the indo-1 fluorescence at 405 nm was independent of changes in [Ca2+]i. B, The rate of fluorescence decrease (or quench rate) at 405 nm was estimated from the slope of a linear fit to the segment of the 405-nm fluorescence in control, in the presence of Mn2+ and in the presence of both Mn2+ and cyanide (CN). The corresponding [Ca2+]i rise due to cyanide is also shown. The result is the average of six similar experiments.

 
Influence of mitochondrial inhibition of depolarization-triggered exocytosis
The above findings suggest that mitochondria play a dominant role in Ca2+ homeostasis of corticotropes. Inhibition of mitochondria prolonged the depolarization-triggered Ca2+ signal (Fig. 3Go). Because activation of voltage-gated Ca2+ entry is the major pathway for CRH-mediated ACTH secretion from corticotropes, we examined how this change in the shape of the Ca2+ signal might affect secretion by monitoring [Ca2+]i simultaneously with exocytosis (membrane capacitance measurement). An example of this experiment is shown in Fig. 7Go. Corticotrope was voltage clamped at –70 mV, and a voltage step was applied to trigger a transient rise in [Ca2+]i before and in the presence of cyanide. Before cyanide application, the peak of the depolarization-triggered [Ca2+]i rise was 3.65 µM, and the cumulative increase in membrane capacitance was 60 femtofarads (fF). Assuming each granule contributed approximately 1.3 fF, the single 500 ms voltage step evoked the release of approximately 46 granules. Application of cyanide raised basal [Ca2+]i by 0.3 µM. In the presence of cyanide, the same voltage step raised [Ca2+]i to 4.1 µM, and the cumulative increase in membrane capacitance was 114 fF (~1.9-fold of the control). Note that in the presence of cyanide, the Ca2+ signal stayed near the peak for several seconds. During this time, the membrane capacitance continued to increase. After the capacitance increase reached a peak, it was followed by a decrease in capacitance (reflecting endocytosis). In contrast, under control condition, the Ca2+ signal started to decay after it had reached its peak, and there was no further increase in membrane capacitance. This result suggests that the cyanide-mediated enhancement in exocytotic response was related to an increase in the amplitude of the depolarization-triggered Ca2+ signal as well as the longer duration at high [Ca2+]i due to the slowing of Ca2+ clearance. In five of six cells examined, the depolarization-triggered exocytotic response was larger in the presence of cyanide. In these five cells, the increase in capacitance by cyanide ranged from 1.4- to 8.9-fold (average = 3.6 ± 1.4-fold), and the increase in the amplitude of the depolarization-triggered Ca2+ signal by cyanide ranged from 0.3–1.4 µM.



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  		FIG. 7. Mitochondrial inhibition enhanced depolarization-triggered exocytosis. Simultaneous measurements of [Ca2+]i and exocytosis (membrane capacitance measurement). The depolarization-triggered [Ca2+]i rise was accompanied by increases in {Delta}Cm, reflecting exocytosis. In the presence of cyanide, the basal [Ca2+]i rose to 0.5 µM and the same depolarization triggered a larger and longer Ca2+ transient. Note that in the presence of cyanide, the increase in {Delta}Cm was followed by a rapid decrease, reflecting endocytosis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results show that in rat corticotropes, after a depolarization-triggered [Ca2+]i rise, the time course of the decay of the Ca2+ signal (with peak [Ca2+]i ranging from 0.7–2 µM) could be well described by a single exponential function with a time constant of 5–6 sec. This kinetic of Ca2+ clearance is different from that reported in gonadotropes (16) and adrenal chromaffin cells (36), where the decay of the Ca2+ signal (with peak [Ca2+]i > 0.5 µM) was biphasic or triphasic. The rate of cytosolic Ca2+ clearance in corticotropes was also considerably slower than that in pancreatic ß-cells [time constant of 1–2 sec (12)] but comparable to melanotropes (37). Among the cytosolic Ca2+ clearance mechanisms, neither NCX nor PMCA has any significant contribution to the rate of cytosolic Ca2+ clearance in corticotropes. Inhibition of SERCA pump in corticotropes resulted in slowing of cytosolic Ca2+ clearance (~1.2-fold), but the magnitude was much smaller than the 3- to 4-fold slowing reported in pancreatic ß-cells and gonadotropes (12, 16). In contrast, inhibition of mitochondrial function slowed the rate of cytosolic Ca2+ clearance in corticotropes by 2- to 3-fold. Thus, mitochondria are the major contributors to cytosolic Ca2+ clearance in rat corticotropes. A dominant role of mitochondria in cytosolic Ca2+ clearance has also been reported in adrenal chromaffin cells (36). The cyanide-mediated slowing of cytosolic Ca2+ clearance in corticotropes could be mimicked by intracellular dialysis of ruthenium red, an inhibitor of mitochondrial uniporter (Fig. 4DGo), suggesting that during depolarization, Ca2+ was rapidly taken up into the mitochondria via the uniporter. This was further confirmed by our rhod-2 fluorescence measurement, which showed a robust increase in mitochondrial Ca2+ signal during depolarization (Fig. 4CGo). In addition, cyanide (Fig. 4AGo) or ruthenium red (Fig. 4BGo) could largely abolish this depolarization-mediated increase in mitochondrial Ca2+ signal. The small contribution of the PMCA to cytosolic Ca2+ clearance in our finding is in contrast to that reported in the AtT-20 cells (17). One possible explanation is the difference between a primary corticotropes and a clonal cell line. On the other hand, the identified corticotropes in the current study were kept in a culture medium containing 10% horse serum for 2–4 d before recording. A recent study in rat hippocampus has shown that PMCA isoform 1 gene expression is repressed by corticosterone (38). Thus, it raises the possibility that the corticosterone present in the serum in our culture medium may reduce the activity of PMCA in cultured corticotropes. Although the concentration of corticosterone in the 10% horse serum in our culture medium is unclear, the AtT-20 cells in which a dominant action of PMCA was reported were cultured with 10% horse serum (17). Others have also reported that corticosterone or dexamethasone (100 nM) could cause robust negative-feedback inhibition of ACTH release from rat pituitary cells, which were cultured with 10% fetal calf serum for 5 d (39, 40). Because the negative-feedback action of glucocorticoid could still be observed in cultured pituitary cells and corticotropes in vivo are exposed to glucocorticoid, we suggest that it is unlikely that the level of glucocorticoid in our culture medium is high enough to cause a major down-regulation of the PMCA in our cultured corticotropes.

We found that in addition to the slowing of Ca2+ clearance, cyanide also caused a rise in basal [Ca2+]i in corticotropes. This rise in basal [Ca2+]i was largely dependent on extracellular Ca2+ influx (Fig. 5AGo) but was not associated with any major increase in membrane current (Fig. 5BGo). Moreover, the result from our Mn2+ quench experiment suggests that cyanide did not activate any additional Ca2+-influx pathway. Interestingly, the Mn2+ quench experiment reveals the presence of a basal Ca2+-influx pathway in rat corticotropes. Under control condition, the basal Ca2+ influx was not associated with any change in basal [Ca2+]i, suggesting that this Ca2+ influx was balanced by Ca2+ uptake into intracellular compartments as well as Ca2+ efflux to the outside of the cell. The presence of such basal Ca2+ influx in corticotropes might underlie the reduction in basal [Ca2+]i (~0.13 µM) when Ca2+ was removed from the extracellular solution. In comparison, the removal of extracellular Ca2+ decreased the basal [Ca2+]i by only 34 nM in rat gonadotropes (16). A portion of the Ca2+ entry via this basal influx pathway in corticotropes is probably taken up by the mitochondria. When mitochondrial Ca2+ uptake was inhibited by cyanide, the basal Ca2+ influx was no longer balanced and, thus, resulted in a gradual elevation of basal [Ca2+]i. At first glance, the involvement of mitochondrial Ca2+ uptake in regulating basal [Ca2+]i appears unlikely because of the low Ca2+ affinity of the mitochondrial uniporter. However, the ability of the mitochondria to rapidly uptake Ca2+ during a short depolarization (500 msec) suggests that in corticotropes, some mitochondria are in close proximity to the plasma membrane. During continuous basal Ca2+ influx, it is likely that the mitochondria near the plasma membrane experience a local high concentration of Ca2+, which activates the uniporter. Consistent with this, inhibition of the mitochondrial uniporter by ruthenium red (10 µM) after approximately 7 min of dialysis resulted in a gradual elevation of basal [Ca2+]i (~0.2 µM) in corticotropes. A recent study has shown that in some cells, the endoplasmic reticulum near the plasma membrane is responsible for transferring extracellular Ca2+ influx to the mitochondria (41). Whether a similar transfer of Ca2+ from endoplasmic reticulum to mitochondria also occurs in corticotropes is unclear. Nevertheless, our observation that basal [Ca2+]i in corticotrope was elevated during inhibition of SERCA or PMCA pumps suggest that these two Ca2+-ATPases have important contribution to the maintenance of basal [Ca2+]i. Thus, in control condition, the basal Ca2+ influx in corticotropes is balanced by the combined actions of the mitochondrial uniporter and SERCA and PMCA pumps. We found that the effect of cyanide on basal [Ca2+]i was reduced from 0.75 to 0.28 µM when the intracellular ATP level was increased from 2–10 mM. Thus, in cells recorded with 2 mM intracellular ATP, a large fraction of the effect of cyanide on basal [Ca2+]i might be attributed to the disruption of mitochondrial ATP production by cyanide. Recent studies suggest that mitochondria are closely associated with the endoplasmic reticulum (42). Therefore, a decrease in the local production of ATP near the PMCA and SERCA pump may reduce their activities, resulting in an imbalance of basal Ca2+ influx and efflux. Interestingly, the slowing of the decay of the depolarization-triggered Ca2+ transient by cyanide was not significantly affected by increasing the intracellular ATP level. This suggests that the mitochondrial Ca2+ uptake is the dominant cytosolic Ca2+ clearance mechanism after a transient [Ca2+]i rise. On the other hand, PMCA and SERCA pump are important in maintaining the basal [Ca2+]i in corticotropes.

This study also shows that mitochondrial inhibition results in an enhancement of exocytotic response in corticotropes (Fig. 7Go). The increase in depolarization-triggered exocytosis was mainly associated with the increase in the duration of the Ca2+ signal as well as the amplitude of the Ca2+ transient. The increase in exocytotic response in the presence of cyanide is probably underestimated here as the prolonged Ca2+ signal frequently triggered fast endocytosis (three of five cells), thus masking any further increase in membrane capacitance. In summary, mitochondrial Ca2+ uptake plays an important role in the regulation of basal [Ca2+]i as well as the shaping of the depolarization-triggered Ca2+ signal in corticotropes. Because the major ACTH secretagogue CRH triggers [Ca2+]i elevation via depolarization and extracellular Ca2+ entry, the uptake of Ca2+ into the mitochondria during CRH stimulation can, in turn, activate mitochondrial metabolism, thus matching the increase in metabolic demand imposed by the secretory activity of the cell. This effect may be particularly important during the diurnal ACTH surge when there is prolonged ACTH secretion from corticotropes. On the other hand, the important role of mitochondria in regulating both basal [Ca2+]i and the depolarization-triggered Ca2+ signal may render the corticotropes particularly sensitive to oxidative and metabolic stress. The intertwined roles of the mitochondria in [Ca2+]i homeostasis and metabolism in rat corticotropes underscore the general importance of mitochondria in the function of corticotropes.


   Acknowledgments
 
We thank Dr. Frederick Tse for comments on the manuscript and Dr. R. J. Kemppainen for the gift of ACTH antibodies.


   Footnotes
 
This work was supported by grants from the Canadian Institute of Health Research and the Alberta Heritage Foundation for Medical Research (AHFMR). A.T. is an AHFMR Senior Scholar.

First Published Online August 4, 2005

Abbreviations: AM, Acetoxymethyl ester; AVP, arginine vasopressin; BHQ, 2,5-di(tert-butyl)-1,4-benzohydroquinone; [Ca2+]i, intracellular Ca2+ concentration; CCCP, carbonyl cyanide m-chlorophenylhydrazone; fF, femtofarad(s); IP3, inositol trisphosphate; NCX, Na+/Ca2+ exchanger; PMCA, plasma membrane Ca2+-ATPase; SERCA, sarco-endoplasmic reticulum Ca2+-ATPase.

Received March 28, 2005.

Accepted for publication July 25, 2005.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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