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Arginine Vasopressin Triggers Intracellular Calcium Release, a Calcium-Activated Potassium Current and Exocytosis in Identified Rat Corticotropes¹

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

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

	Abstract

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Arginine vasopressin (AVP) stimulates the secretion of ACTH from pituitary corticotropes. We investigated the action of AVP in single corticotropes of male rats. Corticotropes were identified with the reverse hemolytic plaque assay using antibodies against ACTH. Using the whole-cell recording technique in conjunction with the fluorescent Ca²⁺ indicator, indo-1 to measure the concentration of cytosolic free Ca²⁺ ([Ca²⁺]_i), we show that AVP triggers a transient and plateau pattern of Ca²⁺ signal. The [Ca²⁺]_i elevation activates the apamin-sensitive Ca²⁺-activated K⁺ current, which, in turn, causes membrane hyperpolarization. The Ca²⁺ signal can be elicited in the absence of extracellular Ca²⁺ and is mimicked by intracellular inositol 1,4,5-trisphosphate (IP₃). Both GDP-ß-S and heparin inhibit the AVP response. Thus, AVP triggers intracellular Ca²⁺ release from the (IP₃)-sensitive store via a GTP binding protein-coupled phosphoinositide pathway. Using the high temporal resolution capacitance measurement to detect exocytosis in single corticotropes, we show that a burst of exocytosis is evoked during the AVP-triggered [Ca²⁺]_i elevation. Exocytosis can also be triggered when Ca²⁺ is released directly from the IP₃-sensitive store via flash photolysis of caged IP₃. We conclude that AVP-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca²⁺ release from the IP₃-sensitive store.

	Introduction

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SECRETION of ACTH from the anterior pituitary gland is stimulated by multiple hypothalamic hormones, including CRH and arginine vasopressin (AVP). CRH is the most efficacious secretagogue, and it evokes a monophasic ACTH secretion via the activation of cAMP-dependent protein kinase A (1). In contrast, AVP elicits transient and plateau phases of ACTH secretion (2). Biochemical studies have shown that AVP receptors in the anterior pituitary gland are coupled to the phospholipase C pathway (3, 4). However, detail understanding of the mechanism underlying the AVP-triggered ACTH secretion has been hindered by the difficulty in identifying corticotropes in the heterogeneous population of pituitary cells, as well as the low temporal resolution of traditional assays for ACTH secretion. In human pituitary adenoma cells, AVP was reported to increase the duration and amplitude of action potential firings, as well as potentiating the L-type voltage-gated Ca²⁺ current (5). In rat pituitary cells, protein kinase C (PKC) was postulated to be essential for the AVP-evoked ACTH secretion (6, 7, 8, 9). Other studies suggested that the transient phase of AVP-evoked ACTH secretion involved intracellular Ca²⁺ release (2, 10), but the plateau phase involved a PKC-mediated extracellular Ca²⁺ entry via L-type voltage-gated Ca²⁺ channels (2, 11). A biphasic pattern of Ca²⁺ signal induced by AVP has also been reported in rat pituitary corticotropes (12, 13, 14). Here, we described the signaling pathway underlying the AVP-triggered Ca²⁺ signal and the role of the Ca²⁺ signal in triggering secretion in single identified rat pituitary corticotropes. An abstract describing some of the results has been published (15).

	Materials and Methods

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Cell identification and culture
The anterior lobe of the pituitary gland was removed from male Sprague-Dawley rats (35–45 days old), killed with halothane, in accordance with the standards of the Canadian Council on Animal Care. Anterior pituitary glands were dissociated enzymatically using collagenase and trypsin, as previously described (16). Single corticotropes were identified from the anterior pituitary cell population by a reverse hemolytic plaque assay (17). The procedures were similar to that described previously for gonadotropes (16, 18). Briefly, the dissociated pituitary cells were suspended in DMEM (Gibco, Grand Island, NY) that contained 0.1% BSA (Sigma, St. Louis, MO). The pituitary cell suspension was mixed with an equal volume of 12% sheep erythrocytes (CO Serum Co., Denver, CO) in 0.9% NaCl. The erythrocytes were previously conjugated with Staphylococcus aureus-derived protein A (Sigma) using 0.2 mg/ml CrCl₃ as catalyst. The cell mixture was incubated with 100 nM CRH (Peninsula Laboratories, Belmont, CA) and rabbit polyclonal antibodies to rat ACTH (1:20 dilution; gift from Dr. Robert J Kemppainen, Auburn University, Auburn, AL) for 3 h at 37 C. Plaques were formed by a 30-min exposure to guinea pig complement (Gibco) at 1:50 dilution. Cells were maintained under standard culture conditions in a DMEM medium supplemented with 10% horse serum (Gibco), 50 U ml^-1 penicillin G (Gibco), and 50 µg ml^-1 streptomycin (Gibco). Recordings were performed on cells maintained in culture for 2–4 days after plaque formation.

Electrophysiological recording
Membrane currents or potentials were recorded with the whole-cell, gigaseal method (19), using an EPC-7 or EPC-9 patch clamp amplifier. Changes in membrane capacitance (C_m) were measured with a dual-phase lock-in amplifier by superimposing an 800-Hz sinusoid of 30-mV peak-to-peak amplitude onto the holding potential, as described previously (20). The value of C_m was low-pass filtered with a register-capacitor circuit of 10 msec time constant. Currents, membrane potential, [Ca²⁺]_i, and C_m values were first recorded on video cassette recorder tapes with a NeuroData PCM recorder, Neuro Data Corp. (New York, NY) and digitized later. The pipettes were made from hematocrit glass (VWR Scientific, London, Ontario, Canada), and the resistance was 2–4 Mohms after filling, and 5–15 Mohms during whole-cell recording. Unless otherwise stated, recordings were done at room temperature (22–25 C). A junction potential correction of -10 mV was applied throughout. Values given in the text are mean ± SEM.

Solutions
The normal external saline solution contained (in mM): 150 NaCl, 10 Na-HEPES, 8 glucose, 2.5 KCl, 2 CaCl₂, and 1 MgCl₂ (pH 7.4). In the Ca²⁺-free saline, CaCl₂ was replaced with MgCl₂, and 1 mM EGTA was added. The standard pipette solution contained (in mM): 120 K-aspartate, 20 KCl, 20 K-HEPES, 2 MgCl₂, 2 Na₂ATP, 0.1 Na₄GTP (pH 7.4).

Heparin, inositol 1,4,5-trisphosphate (IP₃), caged IP₃, and guanosine-5'-O-(2-thiodiphosphate) (GDP-ß-S) were obtained from Calbiochem (La Jolla, CA). The fluorescent Ca²⁺ indicator (indo-1; Calbiochem) was kept as stock solution in distilled water at -20 C. CRH and AVP (Peninsula) were dissolved in 0.1 M acetic acid, lyophilized, and kept at -20 C. The bath was continuously perifused with control or drug solution. The time for a complete exchange of bath solution was approximately 60 sec.

Measurement of [Ca²⁺]_i
[Ca²⁺]_i was measured fluorometrically using the Ca²⁺ indicator, indo-1 (100 µM), dialyzed into the cell via the whole-cell patch pipette. Details of the instrumentation and procedures of the [Ca²⁺]_i measurement were as described previously (16, 21). Briefly, indo-1, in single corticotrope, was excited by 365-nm (band-pass filtered) light delivered from a HBO 100 W mercury lamp via a 40X, 1.3 NA UV fluor oil objective (Nikon Canada, Mississauga, Ontario, Canada). Photon counts were collected at 405 and 500 nm for 20 msec by two photomultiplier tubes (Hamamatsu H3460–04) and then translated into logic signals counted simultaneously by a CYCTM-10 counter card (Cyber Research Inc., Branford, CT) in an IBM-compatible computer. [Ca²⁺]_i was calculated from the ratio (R) of fluorescence at 405 and 500 nm, using the equation of Grynkiewicz, Poenie and Tsien (22), Eq 1: [Ca²⁺]_i = K¹ (R - R_min)/(R_max - R), where R_min is the fluorescence ratio of Ca²⁺-free indo-1 and R_max is the ratio of Ca²⁺-bound indo-1. K¹ is a constant that was determined empirically. Calibrations were determined from groups of single corticotropes (n = 5–8) dialyzed with one of the three pipette solutions, as described previously (21). R_min was measured in cells loaded with (mM): 52 K-aspartate, 10 KCl, 50 K-EGTA, 0.1 indo-1, and 50 K-HEPES (pH 7.4); and R_max was measured in cells loaded with (mM): 136 K-aspartate, 15 CaCl₂, 0.1 indo-1, and 50 K-HEPES (pH 7.4). K¹ was calculated from Eq 1, using R values obtained from cells loaded with (mM): 60 K-aspartate, 50 K-HEPES, 20 K-EGTA, 15 CaCl₂, 0.1 indo-1 (pH 7.4), which had a calculated free Ca²⁺ concentration of 212 nM at 24 C (23). For all experiments reported here, the values of R_min, R_max, and K¹ were 0.468, 5.22, and 2.89 µM, respectively.

	Results

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Electrophysiological effects of AVP on corticotropes
The action of AVP on single rat corticotropes was investigated by simultaneously monitoring [Ca²⁺]_i and membrane current using the voltage clamp technique. The membrane potential of corticotrope was held at -50 mV. Application of AVP (100 nM) triggered a transient rise in [Ca²⁺]_i (Fig. 1). [Ca²⁺]_i rose from the basal level to a peak in approximately 2 sec and then started to decay. The decay comprised a transient phase (in which [Ca²⁺]_i fell to approximately 0.5 µM within approximately 30 sec) and a plateau phase (in which [Ca²⁺]_i lingered as long as AVP was present). Upon the removal of AVP, [Ca²⁺]_i slowly returned to the basal level. Note that the AVP-induced biphasic pattern of Ca²⁺ signal was accompanied by the activation of an outward current (Fig. 1). In six cells examined, the peak [Ca²⁺]_i was 1.58 ± 0.36 µM, and the average peak current amplitude at -50 mV was 56.7 ± 16.5 pA. Application of the bee venom, apamin (0.4 µM), a selective blocker of the small conductance Ca²⁺-activated K⁺ (SK) channels, completely abolished the outward current without affecting the AVP-induced Ca²⁺ signal (Fig. 2; n = 4). Thus, this outward current is a result of the opening of the SK channels during [Ca²⁺]_i elevation. Figure 1 also reveals that the relationship between [Ca²⁺]_i and the activation of Ca²⁺-activated K⁺ current (I_K(Ca)) is complex. When [Ca²⁺]_i rose beyond approximately 0.3 µM, I_K(Ca) was activated. Initially, the current amplitude increased with the rise of [Ca²⁺]_i. However, I_K(Ca) continued to increase (typically for tens of seconds), even after [Ca²⁺]_i had reached its peak. I_K(Ca) remained near its maximum when [Ca²⁺]_i had decayed from its peak to approximately 0.7 µM. A similar discrepancy between [Ca²⁺]_i and I_K(Ca) was observed in all six cells examined.

View larger version (20K): [in this window] [in a new window]   Figure 1. AVP triggered [Ca2+]i elevation and activation of an outward current. Simultaneous measurement of [Ca2+]i and current, in a single corticotrope. The cell was voltage clamped at -50 mV. AVP was bath applied at the time indicated by the bar. The delay in the onset of Ca2+ response is caused mostly by the time required for solution exchange. The time indicated was the time after establishment of whole-cell configuration.

View larger version (22K): [in this window] [in a new window]   Figure 2. The AVP-induced outward current is inhibited by apamin. In the presence of 0.4 µM apamin, AVP elicited [Ca2+]i elevation without activating any outward current. The cell was held at -50 mV.

View larger version (20K): [in this window] [in a new window]   Figure 3. AVP triggered [Ca2+]i elevation and membrane hyperpolarization. Simultaneous measurement of [Ca2+]i and membrane potential in a single corticotrope. Note that the cell was hyperpolarized in both the transient and the plateau phase of the Ca2+ signal.

We further examined the role of extracellular Ca²⁺ in the AVP-induced Ca²⁺ signal. In the experiment shown in Fig. 4, the cell was voltage clamped at -90 mV. Removal of extracellular Ca²⁺ resulted in a reduction of the resting [Ca²⁺]_i by 59 ± 6 nM, and the mean resting [Ca²⁺]_i was 0.13 ± 0.02 µM (n = 8), suggesting that there is a constant passive Ca²⁺ leak across the plasma membrane in unstimulated corticotropes. In 8/10 cells examined, AVP still triggered a biphasic pattern of Ca²⁺ signal in the absence of extracellular Ca²⁺ (Fig. 4). In the remaining two cells, AVP triggered a small transient [Ca²⁺]_i elevation (0.69 ± 0.12 µM), which was not followed by a plateau phase. In the eight cells that exhibited a biphasic pattern of AVP-induced Ca²⁺ signal in the absence of extracellular Ca²⁺, [Ca²⁺]_i was 2.32 ± 0.30 µM during the transient phase and declined to 0.21 ± 0.03 µM during the plateau phase. Considering the lower resting [Ca²⁺]_i (0.06 µM smaller) of cells bathed in Ca²⁺-free saline, the [Ca²⁺]_i levels during the transient and the plateau phases were still slightly lower than those obtained in the presence of 2 mM extracellular Ca²⁺ (2.54 ± 0.28 and 0.31 ± 0.04 µM, respectively). However, the rate of decay of [Ca²⁺]_i during the plateau phase was not affected by the absence of extracellular Ca²⁺. Between 100–120 sec after the peak of [Ca²⁺]_i elevation, the decay of Ca²⁺ was negligible (-0.21 ± 0.18 nM/sec; n = 8). These results suggest that there is some loss of Ca²⁺ from intracellular stores in the absence of extracellular Ca²⁺. Nevertheless, the presence of both the transient phase and plateau phase in the AVP-induced Ca²⁺ signal of the majority of cells (8 of 10) bathed in Ca²⁺-free solution indicates that intracellular Ca²⁺ release contributes significantly to both phases of the Ca²⁺ signal.

View larger version (15K): [in this window] [in a new window]   Figure 4. In the absence of extracellular Ca2+, AVP can trigger both the transient phase and the plateau phase of Ca2+ signal elevation. At the time indicated, the external saline solution was switched to a Ca2+-free saline, which contained 1 mM EGTA. The cell was held at -90 mV.

View larger version (17K): [in this window] [in a new window]   Figure 5. AVP triggered intracellular Ca2+ release via a G-protein-coupled phosphoinositide pathway. A, AVP fails to induce [Ca2+]i elevation when activation of G-protein is prevented by GDP-ß-S. The pipette solution contained 2 mM GDP-ß-S. B, Intracellular IP3 triggers [Ca2+]i elevation. The pipette solution contained 10 µM IP3, and the recording started at approximately 10 sec after establishment of whole-cell configuration. Subsequent AVP application failed to elicit [Ca2+]i elevation, implicating the depletion of the intracellular store. C, AVP fails to trigger [Ca2+]i elevation in the presence of the competitive blocker of IP3 receptor, heparin. The pipette solution contained 100 µM heparin. Cells were held at -50 mV in all experiments.

View larger version (23K): [in this window] [in a new window]   Figure 6. The AVP-induced [Ca2+]i elevation is accompanied by exocytosis. Simultaneous measurement of [Ca2+]i and changes in membrane capacitance (Cm). The cell was voltage clamped at -70 mV. The external saline contained 0.5 µM apamin, to inhibit the Ca2+-activated K+ current.

View larger version (20K): [in this window] [in a new window]   Figure 7. Intracellular Ca2+ release from the IP3-sensitive store is sufficient to trigger exocytosis. The pipette solution contained 10 µM caged IP3, and the cell was voltage clamped at -70 mV. A UV flash was delivered at the time indicated by the arrow.

In all experiments involving flash photolysis of caged IP₃ (e.g. Fig. 7), [Ca²⁺]_i rose rapidly to its peak and remained near the maximum for approximately 2 sec, before starting to decline. Exocytosis typically started to occur when [Ca²⁺]_i had almost reached its peak and the minimum [Ca²⁺]_i required to trigger exocytosis was 1.80 ± 0.16 µM (n = 8). Note that in these cells (e.g. Fig. 7), the rise in membrane capacitance continued for approximately 1 sec before starting to decrease, reflecting endocytosis. Within 10–20 sec, the membrane capacitance was gradually restored to its initial value. Because the rate of endocytosis seems to be rather slow during the initial rising phase of the capacitance change (C_m), it is reasonable to assume that exocytosis triggered within 1 sec after the flash was not significantly contaminated by endocytosis. Based on this assumption, the rate of exocytosis at peak [Ca²⁺]_i can be estimated by measuring the average increase in capacitance (during a 0.3–0.5-sec interval) after [Ca²⁺]_i had reached its peak. In eight cells examined, the mean rate of exocytosis was 100 ± 28 fF/s when [Ca²⁺]_i was 1.98 ± 0.09 µM.

	Discussion

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The biphasic pattern of AVP-induced Ca²⁺ signal
Our results demonstrate that AVP stimulates intracellular Ca²⁺ release from the IP₃-sensitive stores in corticotropes via the G-protein-coupled phosphoinositide pathway. Consistent with previous studies (12, 13, 14), AVP triggered a biphasic pattern of Ca²⁺ signal in corticotropes (Fig. 1). The literature has suggested that the transient phase of the AVP-triggered Ca²⁺ signal is caused by intracellular Ca²⁺ release, but the plateau phase requires extracellular Ca²⁺ entry via L-type Ca²⁺ channels (12, 13, 14). However, our data show that the plateau phase (2 min after the transient phase) of AVP-induced [Ca²⁺]_i elevation was still present when corticotropes were voltage clamped at -90 mV to prevent Ca²⁺ entry via voltage-gated Ca²⁺ channels or when extracellular Ca²⁺ was omitted (Fig. 4). Our simultaneous measurements of [Ca²⁺]_i and current show that an apamin-sensitive I_K(Ca) is activated during the plateau phase of AVP-induced Ca²⁺ signal (Fig. 1). Although the amplitude of the current was small, it caused significant membrane hyperpolarization (Fig. 3) because of the high resting input resistance of corticotropes. Because voltage gated Ca²⁺ channels became activated at approximately -50 mV (24), it was unlikely that extracellular Ca²⁺ entry via voltage-gated Ca²⁺ channels was essential for the generation of the plateau phase of the Ca²⁺ signal. What gives rise to the biphasic pattern of Ca²⁺ signal in corticotropes is unclear. It is possible that the replenishment of the IP₃-sensitive stores in corticotropes is slow, such that the majority of the stores are depleted of calcium by the time [Ca²⁺]_i reaches its peak. Later, with a smaller amount of Ca²⁺ being released, the Ca²⁺ removal mechanism becomes dominant and [Ca²⁺]_i starts to fall. Gradually a portion of the stores is replenished, and during the plateau phase, Ca²⁺ removal is balanced by Ca²⁺ release. Alternatively, the biphasic pattern may involve Ca²⁺ removal with complex kinetics. In gonadotropes (21), when the depolarization-induced [Ca²⁺]_i elevation was more than 0.5 µM, [Ca²⁺]_i decayed rapidly (<5 sec) to a plateau of approximately 0.2 µM and lingered at this level for approximately 10 sec, before returning to the basal level. This plateau was postulated to involve mechanisms such as a sudden shutdown of the dominant transport mechanisms or an additional dumping of Ca²⁺ from an intracellular pool into the cytoplasm (21). In chromaffin cells (28), export of Ca²⁺ from mitochondria was shown to contribute to the plateau phase of Ca²⁺ decay. Whether corticotropes may have a similar Ca²⁺ removal kinetics awaits further experiments.

Relationship between AVP-induced [Ca²⁺]_i elevation and I_K(Ca)
The AVP-induced [Ca²⁺]_i elevation opened the SK-type Ca²⁺-activated K⁺ channels and resulted in an outward current (Fig. 1). During the rising phase of the Ca²⁺ signal, I_K(Ca) became detectable at [Ca²⁺]_i beyond 0.3 µM. Before [Ca²⁺]_i reached approximately 1 µM, the current amplitude increased with the rise of [Ca²⁺]_i. However, when [Ca²⁺]_i rose from 1 to 1.5 µM, the Ca²⁺ rise was not accompanied by any further increase in I_K(Ca), suggesting that I_K(Ca) might be saturated at [Ca²⁺]_i > 1 µM. This Ca²⁺-sensitivity of I_K(Ca), during the rising phase of the Ca²⁺ signal, closely resembled the SK channels in pituitary gonadotropes (29), where I_K(Ca) became detectable at [Ca²⁺]_i > 0.3 µM and reached a maximum at [Ca²⁺]_i > 1.2 µM. Similar Ca²⁺ sensitivity has also been reported in cultured rat skeletal muscle (30) and rat chromaffin cells (31), where [Ca²⁺]_i of 0.35 and 0.7 µM were required, respectively, for half-maximal activation of SK channels. However, the relationship between I_K(Ca) and [Ca²⁺]_i is more complex during the decay phase of the Ca²⁺ signal in corticotropes. After [Ca²⁺]_i had reached its peak and started to decay, there was a gradual enhancement of I_K(Ca) (Fig. 1). I_K(Ca) continued to increase for tens of seconds after [Ca²⁺]_i had reached its maximum. The current started to decrease when [Ca²⁺]_i fell to approximately 0.7 µM. What gives rise to this enhancement of I_K(Ca) during the decay of the Ca²⁺ signal is unclear. In rat gonadotropes, IP₃ may release Ca²⁺ selectively from subsurface cisternae, such that a local Ca²⁺ gradient is created near the plasma membrane (32). Because our experiments in corticotropes monitor only the average cytosolic free Ca²⁺ [Ca²⁺]_i, it is possible that during the decay of the Ca²⁺ signal, only the IP₃-sensitive stores near the plasma membrane continue to release Ca²⁺, such that a local spatial Ca²⁺ gradient is generated near the plasma membrane. Under such circumstances, the local [Ca²⁺] levels near the SK channels are higher than the average [Ca²⁺]_i reported by indo-1. Another possible explanation is that the activation of PKC during AVP receptor stimulation may enhance I_K(Ca) by acting directly on the SK channels. In pituitary gonadotropes, PKC augments the maximum amplitude of I_K(Ca) at a wide range of [Ca²⁺]_i (0.3–1.5 µM) and also reduces the [Ca²⁺]_i at which peak I_K(Ca) is half-maximum by 20–30% (29). If PKC has a similar action in corticotropes, the reduction of I_K(Ca) caused by the decay of [Ca²⁺]_i could be counterbalanced by the PKC mediated-I_K(Ca) enhancement.

Exocytosis triggered by AVP or IP₃
Our simultaneous measurement of [Ca²⁺]_i and exocytosis demonstrates that exocytosis is triggered during the AVP-induced [Ca²⁺]_i elevation in corticotropes (Fig. 6). The average cumulative increase in membrane capacitance during a single AVP challenge was approximately 50 fF, corresponding to an exocytosis of approximately 37 granules. The activation of PKC does not seem to be essential for the initial trigger of exocytosis, because exocytosis can be triggered when Ca²⁺ is directly released from the IP₃-sensitive stores via flash photolysis of caged IP₃ (Fig. 7). On average, flash photolysis of caged IP₃ triggered a larger and more rapid increase in capacitance than AVP application, probably because of a larger jump in IP₃ concentration immediately after the flash photolysis of caged IP₃. Whether PKC activation is important for the plateau phase of secretion (>3 min) in corticotropes cannot be addressed here. In our experiments, the increase in membrane capacitance during AVP typically stopped in less than 30 sec (Fig. 6). However, the amount of secretion occurring in the plateau phase may be small. In perifused rat pituitary cells, the amount of ACTH secreted during the plateau phase of AVP stimulation was only approximately 10–15% of the transient phase (2). If a similar percentage of secretion occurs during the plateau phase of AVP stimulation in our experiments (>30s), only approximately 7 granules (10 fF) per minute will be exocytosed. Because the capacitance measurement monitors changes in membrane surface area, it is likely that such small and slow exocytosis will be masked by endocytosis.

Nevertheless, the fast elevation of [Ca²⁺]_i and the rapid increase in capacitance during flash photolysis of caged IP₃ allowed us to estimate the rate of exocytosis in corticotropes without significant contamination from endocytosis. When exocytosis in corticotropes was compared with pituitary gonadotropes (32), in which flash photolysis of caged IP₃ elicited a similar rate of rise of [Ca²⁺]_i, interesting differences were observed. In gonadotropes, exocytosis started to occur, at [Ca²⁺]_i < 0.5 µM, and typically stopped before [Ca²⁺]_i reached its peak (32). In contrast, exocytosis in corticotropes started to occur when [Ca²⁺]_i almost reached its peak, at approximately 1.8 µM. The rate of exocytosis also differed between the two pituitary cell types. In gonadotropes (32), the rate was approximately 250 fF/sec at 1 µM [Ca²⁺]_i, whereas in corticotropes, the rate was approximately 100 fF/sec, even when [Ca²⁺]_i was approximately 2 µM. However, these values must be taken with caution, because in gonadotropes, a local Ca²⁺ gradient is created near the plasma membrane during IP₃-triggered intracellular Ca²⁺ release, such that [Ca²⁺] near the exocytic sites can be 5-fold above the cell average (32). Whether such a local Ca²⁺ gradient is present in corticotropes will require a detail comparison of the Ca²⁺ dependence of the rate of exocytosis using caged Ca²⁺ compounds.

	Acknowledgments

 
We thank Dr. Robert J. Kemppainen for the ACTH antibodies and Dr. Frederick W. Tse for commenting on the manuscript.

	Footnotes

 
¹ This work is supported by grants from the Canadian Medical Research Council and the Alberta Heritage Foundation for Medical Research.

Received October 6, 1997.

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