Effect of Ca2+ and cAMP on capacitance-measured hormone secretion in human GH-secreting adenoma cells

Tsukasa Takei, Junko Yasufuku-Takano, Koji Takano, Toshiro Fujita, and Naohide Yamashita

Fourth Department of Internal Medicine, Tokyo University Branch Hospital, Tokyo 112, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Membrane capacitance (Cm) was measured as an index of exocytosis in human growth hormone-secreting adenoma cells using the perforated whole cell, patch-clamp technique; the effects of membrane depolarization, growth hormone-releasing hormone, and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) were examined. Cm was increased by membrane depolarization to potentials beyond the threshold necessary to open voltage-gated Ca2+ channels. These voltage-dependent changes in Cm varied as a function of both depolarization amplitude and duration and were blocked in the presence of the Ca2+ channel antagonist nitrendipine (10-6 M). When membrane potential was clamped at the holding potential (-78 mV), voltage-gated Ca2+ channels were closed, and neither application of growth hormone-releasing hormone nor 8-BrcAMP affected Cm. However, when these agents were applied to depolarized cells, where the voltage-gated Ca2+ channels were open, the increases in Cm were augmented. From these data, it was concluded that elevation of intracellular cAMP, per se, did not stimulate exocytosis. Rather, Ca2+ influx through voltage-gated channels was a prerequisite for cAMP-induced exocytosis.

growth hormone-releasing hormone; 8-bromoadenosine 3',5'-cyclic monophosphate; nitrendipine

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

SECRETION OF GROWTH HORMONE (GH) from the anterior pituitary gland is stimulated by the hypothalamic peptide GH-releasing hormone (GHRH) (28). GHRH depolarizes the plasma membranes of GH-secreting cells by activating nonselective cation channels (5, 15, 19, 25) and by enlarging voltage-gated Ca2+ channel currents (4, 19, 26). Both of these changes of ion channel behavior are mediated by increases in the levels of the intracellular second messenger, cAMP (2). It is known that the membrane depolarization and augmented Ca2+ currents elicited by GHRH facilitate Ca2+ influx, thereby stimulating GH secretion. However, the precise functional roles played by Ca2+ and cAMP during secretion of GH have not yet been clarified because it has proven difficult to selectively resolve their respective actions. In pancreatic beta -cells and lactotrophs, direct action of cAMP on exocytosis has been reported (1, 8, 24). However, it remains unclear whether this cAMP-induced exocytosis is exclusively independent of Ca2+.

In the present study, we examined the respective effects of Ca2+ and cAMP on GH secretion in human GH-secreting adenoma cells. To accomplish this, membrane capacitance (Cm) was measured as an index of exocytosis (17). We used the perforated whole cell clamp technique to record Cm (9, 11) because it eliminates problems associated with washout of intracellular substrates. In addition, evoked changes in Cm differ depending on whether they are recorded with the use of perforated patch or conventional whole cell clamp techniques (22); for measurement of exocytosis under the physiological conditions, perforated whole cell clamp is preferable. Using this technique, we were able to clarify the respective roles of Ca2+ influx and cAMP in the secretion of GH from single cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell preparation and culture. GH-secreting pituitary adenomas were obtained from three acromegalic patients by transsphenoidal surgery. The Ethical Committee of Tokyo University School of Medicine permits the use of human pituitary tissue obtained at the surgery for experimental purposes. The adenomas were minced into small pieces (<1 mm) and digested with 1,000 U/ml Dispase (protease). Cells to be used in the analysis of hormone release were seeded into 24-well dishes at a density of 1 × 105 cells/dish, and cells to be used in electrophysiological experiments were seeded into 35-mm culture dishes. The cells were cultured in DMEM containing 10% heat-inactivated FCS without antibiotics and were maintained at 37°C under an atmosphere of humidified air containing 5% CO2. Hormone release studies were carried out after 1-2 wk of culture, and the electrophysiological studies were performed after 1-4 wk of culture. Electrophysiological properties and capacitance-measured exocytosis did not change during this period.

Measurement of GH release. Cells cultured in 24-well dishes were washed twice with serum-free DMEM containing 0.1% BSA. They were then incubated in the serum-free medium for 2 h with or without GHRH. After incubation, the medium was collected and stored at -20°C until it was assayed for GH with the use of an RIA kit (Daiichi Radioisotope Laboratories, Tokyo, Japan). The sensitivity of the assay was 0.1 ng/ml, and the intra- and interassay coefficients of variance were 1.1 and 2.1%, respectively. Basal levels of GH release were 255.8 ± 46.3 ng/ml in adenoma 1, 156.0 ± 43.9 ng/ml in adenoma 2, and 20.9 ± 2.8 ng/ml in adenoma 3 (mean ± SD, n = 4). In the presence of 10-8 M GHRH, release increased to 381.5 ± 39.4 ng/ml in adenoma 1, 791.3 ± 143.4 ng/ml in adenoma 2, and 27.6 ± 3.4 ng/ml in adenoma 3. These increases in GH release were statistically significant when analyzed using Student's t-test (P < 0.05).

Perforated patch, whole cell clamp technique. All experiments were performed at room temperature (22-25°C) while the cells were being continuously superfused with the use of a peristaltic pump (~1.5 ml/min). The various agents employed in the study were applied to the cells by changing the superfusing solution. Cm was analyzed using the perforated whole cell clamp technique (11); details of the technique have been reported elsewhere (29). Briefly, patch electrodes were prepared from 1.5-mm glass capillaries containing filaments. When filled with solution, the resistance of the patch electrodes was 5-8 MOmega . A List EPC-7 amplifier was used for recording membrane currents and potentials. Liquid junction potentials between the extracellular and pipette solutions (-8 to -6 mV) were measured using a 3 M KCl electrode as a reference, and all data were corrected accordingly. When whole cell perforated patch-clamp measurements were made, the patches were perforated using nystatin. Shortly before beginning to record, aliquots of nystatin stock solution (50 mg/ml DMSO), which was prepared daily, were diluted in the pipette solution to a final concentration of 200 µg/ml. Voltage clamp recordings were begun after the series resistance fell below 20 MOmega . Because the amplitudes of the recorded currents were <150 pA, errors caused by the series resistance were ignored. Data were filtered with a bandwidth of 1 kHz, and the sampling frequency was 100 µs.

Measurement of Cm. The methods used for measuring Cm were essentially the same as described by Maruyama (18). Cm was measured using a two-phase lock-in-amplifier (NF5610B, NF Instruments, Yokohama, Japan). Initially, command pulses were applied to voltage-clamped cells from a holding potential of -78 mV, and the transient capacitive surges were offset using the cancellation circuit of the EPC-7 amplifier. Then a 200-Hz, 10-mV peak-to-peak sine wave voltage was superimposed on the holding potential. The resultant current output was fed into the lock-in-amplifier. The phase offset of the lock-in-amplifier was adjusted such that when a capacitance calibration of 1 pF was applied, there was no detectable change in 0-phase output (membrane conductance: Delta G) but maximal changes in pi /2-phase output (membrane capacitance: Delta C). Calibrations for Delta C and Delta G were obtained by applying known Delta C and Delta G signals and rescaling them to fit the Delta C and Delta G scales, respectively. The total cell Cm of human GH-secreting adenoma cells ranged from 6 to 8 pF. During application of GHRH or 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), the depolarized membrane potential was fixed at -18 or -28 mV; representative changes of Delta C and Delta G are shown (see Figs. 2, 4, 6-8).

Cell identification. In early experiments, GH staining was used to identify GH-secreting adenoma cells. Cells (n = 4) that had membranes depolarized by application of GHRH (25) were fixed in 10% formaldehyde and immunohistochemically stained for human GH (hGH) using an hGH immunostaining kit (DACO, Glosprup, Denmark), which uses the rabbit polyclonal antibodies to hGH. All of the GHRH-sensitive cells stained positive for hGH. In subsequent experiments, data were obtained from cells that were presumed to be GH-secreting adenoma cells. Under a phase-contrast microscope, these cells were rounded, had glittering surfaces, and were easily discriminated from fibroblast-like cells.

Solutions and reagents. The standard internal pipette solution contained (in mM) 95 potassium aspartate, 47.5 KCl, 1 MgCl2, 0.1 EGTA [tetramethylammonium (TMA) salt], and 10 HEPES (TMA salt, pH 7.2). The standard external solution was (in mM) 128 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, and 10 HEPES (Na salt, pH 7.4). When Ca2+ channel currents were recorded, K+ in the pipette solution was isosmotically replaced with Cs+, and 25 mM Na+ in the extracellular medium was isosmotically replaced with tetraethylammonium ion. Tetrodotoxin (10-7 M) was also present in the extracellular medium.

Human GHRH, 8-BrcAMP, and nystatin were purchased from Sigma (St. Louis, MO). Dispase was purchased from Godo Shusei (Tokyo, Japan).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Changes in Cm caused by Ca2+ influx through voltage-gated channels. Figure 1, A and B, depicts voltage-gated Ca2+ channel currents recorded in the standard extracellular medium containing 2.5 mM Ca2+ ions. To block outward K+ currents, cells were dialyzed with Cs+ from the patch electrodes, and 25 mM tetraethylammonium ion was present in the extracellular solution. The Ca2+ currents were carried through T- and L-type Ca2+ channels, which is consistent with earlier recordings made using Ba2+ ions as the charge carrier (30). The current-voltage relationships are shown in Fig. 1C. The threshold potential for T-type Ca2+ channels was approximately -50 mV, and for L-type Ca2+ channels, the threshold was shifted by ~10 mV toward more depolarized potentials.


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Fig. 1.   Ca2+ currents in human growth hormone (GH)-secreting adenoma cells. A and B: Ca2+ currents elicited by depolarizing membranes to -36 mV (A) and -16 mV (B) from a holding potential of -76 mV. Patch electrode contained 142.5 mM Cs+, and extracellular solution contained 25 mM tetraethylammonium ion. C: current-voltage relationships for T- and L-type Ca2+ currents. Amplitudes of L-type currents were measured as amplitude of steady current, and amplitudes of T-type currents were estimated by subtracting L-type current amplitude from peak amplitude of total current. Depicted currents were recorded in a cell from adenoma 2.

When cell membranes were depolarized beyond the threshold potential for periods of 10 s, obvious increases in Cm were observed (Fig. 2). These changes in Cm were dependent on the magnitudes of the membrane depolarizations (Figs. 2 and 3); in Fig. 3, the maximal increases in Cm, measured in five representative cells, are plotted as a function of membrane potential. As can be seen, the increments in Cm appeared to correspond to the voltage-dependent changes in the Ca2+ currents (Figs. 1C and 3). After termination of the membrane depolarization, Cm declined, but the rate did not appear to be potential dependent. When the membrane was hyperpolarized from the holding potential of -78 mV, no apparent changes in Cm were seen (data not shown).


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Fig. 2.   Effect of membrane depolarization on membrane capacitance. Holding potential was -78 mV, and depolarizing pulse duration was 10 s. Membrane potentials (V) are shown in bottom trace; time-dependent changes in membrane capacitance (Delta C; top) and conductance (Delta G; middle) are also shown. Broken line indicates basal conductance. Intracellular and extracellular media were standard. Cell was from adenoma 1.


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Fig. 3.   Relationship between membrane depolarization and exocytosis. Amplitude of maximum increase in membrane capacitance (ordinate) is plotted as a function of membrane potential (abscissa). Means ± SD of capacitance increases measured in 5 representative cells are shown; in some data, bars are small and hidden by symbols. Cells were from adenomas 1-3 (n = 2, adenoma 1; n = 2, adenoma 2; and n = 1, adenoma 3).

It was observed that the changes in Cm were also dependent on the duration of the membrane depolarization (Figs. 4 and 5). When the representative cell in Fig. 4 was depolarized to -18 mV from a holding potential of -78 mV, for periods of 1, 5 or 10 s, the amplitudes of the changes in Cm increased as the depolarization became more prolonged. The maximum amplitudes of the increases in Cm in four cells are plotted as a function of depolarizing pulse duration in Fig. 5. As the pulse duration became longer, the amplitudes of Cm increased, until they saturated at a pulse duration of 30 s.


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Fig. 4.   Effect of pulse duration of membrane depolarization on increases in membrane capacitance. Holding potential was -78 mV, and membranes were depolarized to -18 mV. Duration of depolarizing potentials (V) is indicated in bottom trace; Delta C (top) and Delta G (middle) are also shown. Broken line indicates basal level of Delta C. Intracellular and extracellular media were standard. Cell was from adenoma 1.


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Fig. 5.   Relationship between duration of membrane depolarization and changes in membrane capacitance. Amplitude of maximum increase in membrane capacitance (ordinate) is plotted as a function of time (abscissa). Means ± SD of capacitance increases measured in 4 representative cells are shown; in some data, bars are small and hidden by symbols. Cells were from adenoma 2.

These data indicate that voltage-dependent Ca2+ influxes were important for the increases in Cm observed in human GH-secreting adenoma cells. To further explore this notion, we examined the effect of a Ca2+ channel blocker, nitrendipine, on depolarization-induced changes in Cm; it has already been reported that nitrendipine inhibits voltage-gated Ca2+ currents in human GH-secreting adenoma cells (30). Figure 6 shows that in the presence of 10-6 M nitrendipine, membrane depolarization did not increase the Cm. Thus Ca2+ influx through voltage-gated channel is important for exocytosis in human GH-secreting adenoma cells. This experiment also indicates that membrane depolarization does not, per se, evoke exocytosis.


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Fig. 6.   Effect of nitrendipine on membrane depolarization-induced increases in membrane capacitance. Nitrendipine (10-6 M) was added to extracellular medium. Holding potential was -78 mV, and depolarized potential was -18 mV. Delta C and V are shown in top and bottom traces, respectively. Cell was from adenoma 2.

Effect of GHRH and cAMP on the Cm increase. Figure 7A shows the changes of the Cm elicited by application of 10-8 M GHRH. When applied while membrane potential was held at -78 mV, a condition under which voltage-gated Ca2+ channels should be closed (Fig. 1C), GHRH had no effect on Cm. However, after the cell membrane was depolarized to -18 mV, an increase in the Cm was observed, similar to the case of Figs. 2 and 4. To separate the respective effects of GHRH and depolarization, GHRH was applied after the membrane had been depolarized to -18 mV and Cm had plateaued (Fig. 7B). Under these conditions, GHRH elicited further increases in Cm.


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Fig. 7.   Effect of GH-releasing hormone (GHRH) on membrane capacitance. A: GHRH (10-8 M) was applied at holding potential of -78 mV. B: GHRH (10-8 M) was applied after membrane was depolarized to -18 mV from holding potential. Delta C and V are shown in top and bottom traces, respectively. Broken lines indicate basal capacitance before membrane depolarization or application of GHRH. Intracellular and extracellular media were standard. Cells were from adenoma 2.

The biological actions of GHRH are mediated by cAMP (2). Therefore, we next examined the effect of cAMP on the Cm. Figure 8 shows the changes in Cm elicited by application of the membrane-permeable cAMP analog 8-BrcAMP (10-5 M). At the holding potential of -78 mV, Cm was unaffected by 8-BrcAMP (Fig. 8A). On the other hand, after depolarization to -28 mV, 8-BrcAMP elicited a slow rise in Cm that was similar to that seen in the presence of GHRH (Fig. 8B). From these results, it was concluded that GHRH-induced exocytosis was mediated by a rise in intracellular cAMP and that GHRH (cAMP) did not stimulate exocytosis when voltage-gated Ca2+ channels were not activated. Therefore, Ca2+ influx through voltage-gated channels appeared to be a prerequisite for GHRH (cAMP)-induced increases in Cm.


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Fig. 8.   Effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on membrane capacitance. A: 8-BrcAMP (10-5 M) was applied at holding potential of -78 mV. B: 8-BrcAMP (10-5 M) was applied after membrane was depolarized to -28 mV from holding potential. Delta C and V are shown in top and bottom trances, respectively. Intracellular and extracellular media were standard. Cells were from adenoma 2.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The results of the present experiments revealed that Ca2+ influx through voltage-gated Ca2+ channels was essential for capacitance-measured exocytosis in human GH-secreting adenoma cells. The membrane depolarization, which activated voltage-gated Ca2+ channels, stimulated exocytosis, and the application of the Ca2+ channel blocker nitrendipine inhibited it. In human GH-secreting adenoma cells, we have reported that nitrendipine inhibited GH secretion, as measured by RIA (30), and that it also inhibited GHRH-induced increases in the concentration of intracellular Ca2+ ([Ca2+]i) (26). The results of the present study are in agreement with these earlier results.

The amplitudes of the changes in Cm increased with the amplitudes of the evoked Ca2+ currents (Figs. 1C and 3). This suggests that the amount of exocytosis increased in parallel with the increment in Ca2+ influx through voltage-gated channels. These data are consistent with observations made in bovine adrenal chromaffin cells, in which exocytosis was also measured with the use of the perforated whole cell clamp technique (7). In that study, the amount of exocytosis was strictly related to the integral of the voltage-gated Ca2+ current. However, exocytosis tended to saturate when the duration of the membrane depolarization was longer than 30 s (Fig. 5). It has been known that there are two kinds of secretory vesicles, large dense-core and small clearer vesicles; hormone-containing granules in endocrine cells are among the large dense-core vesicles. The dependence of exocytosis on [Ca2+]i is different between large dense-core vesicles and small clearer vesicles (13). Exocytosis is comparatively slow for large dense-core vesicles and requires relatively low [Ca2+]i compared with the small clearer vesicles. It is probable that the increase of [Ca2+]i caused by longer depolarizations was sufficient to saturate the exocytosis process in GH-secreting adenoma cells. A similar case in which prolonged membrane depolarizations saturated exocytosis was observed in rat posterior pituitary cells (12). In GH-secreting adenoma cells, there are two types of voltage-gated Ca2+ channels, T type and L type. Because T-type channels inactivate within 200 ms, L-type channels account for exocytosis in the case of prolonged membrane depolarization. In fact, nitrendipine more effectively inhibits L-type channels (30).

Application of GHRH and 8-BrcAMP did not increase Cm when membrane potentials were more hyperpolarized than the threshold for opening voltage-gated Ca2+ channels. However, these agents augmented depolarization-induced increases in Cm when voltage-gated Ca2+ channels were activated. This indicates that elevation of intracellular cAMP does not, by itself, stimulate exocytosis. Instead, cAMP only exerts its stimulative effect in conjunction with a Ca2+ influx through voltage-gated channels. These results are in agreement with the report that extracellular Ca2+ ions are essential for GHRH-induced GH release in rat anterior pituitaries (2, 14).

A brief membrane depolarization caused an increase in Cm that gradually declined after repolarization (Fig. 2). The time course of the decrease in the Cm fluctuated and did not depend on the magnitude of the brief depolarization. Similar fluctuations in Cm were also observed during prolonged membrane depolarizations (Fig. 7). It has been reported that endocytosis concomitantly occurs during exocytosis and that endocytosis was also regulated by [Ca2+]i (3, 6, 10, 20, 27). Although the mechanisms of endocytosis in human GH-secreting adenoma cells were not examined in the present experiment, it was surmised that the observed fluctuations in Cm were caused by endocytosis.

Previous studies using rat or human GH-secreting cells revealed that GHRH activates nonselective cation channels and, thereby, depolarizes the plasma membrane (5, 15, 19, 25); this membrane depolarization increases the firing of Ca2+-dependent action potentials. GHRH also enhances voltage-gated Ca2+ channel currents (4, 19, 26), facilitating Ca2+ influx and increasing [Ca2+]i. Although increases in [Ca2+]i are implicated in hormone secretion from anterior pituitaries (21), the role of cAMP has not been fully elucidated in somatotrophs. In pancreatic beta -cells, cAMP enhances Ca2+ influx through voltage-gated Ca2+ channels and increases [Ca2+]i in a manner similar to that seen in human GH-secreting adenoma cells (16). This cAMP-induced increase in [Ca2+]i promoted exocytosis, but in beta -cells, cAMP may also stimulate exocytosis that is independent of [Ca2+]i (1, 8). It was suggested that cAMP may directly affect the mechanism of insulin granule mobilization. However, [Ca2+]i-independent insulin release caused by cAMP disappeared when [Ca2+]i was below 60 nM (23). Similarly, it was suggested that cAMP directly facilitates exocytosis in bovine lactotrophs, but this effect was inhibited when [Ca2+]i was chelated with 10 mM EGTA (24). In spontaneously firing human GH-secreting adenoma cells, [Ca2+]i ranged from 150 to 225 nM (26). When Ca2+ influx through voltage-gated channels was inhibited, it decreased below this range, suggesting that cAMP-evoked exocytosis required [Ca2+]i >150 nM in human GH-secreting adenoma cells. Thus it appears that physiological levels of [Ca2+]i are required for cAMP-induced exocytosis, but the level of [Ca2+]i necessary for exocytosis likely differs among cell types. In the case of human GH-secreting adenoma cells, it may be necessary for [Ca2+]i to be higher than in pancreatic beta -cells and bovine lactotrophs.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: N. Yamashita, Dept. of Advanced Medical Science, Institute of Medical Science, Univ. of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Received 29 April 1998; accepted in final form 2 July 1998.

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Abstract
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Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(4):E649-E654
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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