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Am J Physiol Endocrinol Metab 292: E1863-E1870, 2007. First published February 27, 2007; doi:10.1152/ajpendo.00047.2007
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Somatostatin decreases voltage-gated Ca2+ currents in GH3 cells through activation of somatostatin receptor 2

Seung-Kwon Yang,1,2 Helena C. Parkington,2 Jacques Epelbaum,3 Damien J. Keating,1 and Chen Chen1,2

1Prince Henry's Institute of Medical Research, Melbourne, Australia; 2Department of Physiology, Monash University, Melbourne, Australia; and 3UMR 549 INSERM, Faculté de Médecine Université Paris Descartes, Paris, France

Submitted 18 January 2007 ; accepted in final form 22 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The secretion of growth hormone (GH) is inhibited by hypothalamic somatostatin (SRIF) in somatotropes through five subtypes of the somatostatin receptor (SSTR1–SSTR5). We aimed to characterize the subtype(s) of SSTRs involved in the Ca2+ current reduction in GH3 somatotrope cells using specific SSTR subtype agonists. We used nystatin-perforated patch clamp to record voltage-gated Ca2+ currents, using a holding potential of –80 mV in the presence of K+ and Na+ channel blockers. We first established the presence of T-, L-, N-, and P/Q-type Ca2+ currents in GH3 cells using a variety of channel blockers (Ni+, nifedipine, {omega}-conotoxin GVIA, and {omega}-agatoxin IVA). SRIF (200 nM) reduced L- and N-type but not T- or P/Q-type currents in GH3 cells. A range of concentrations of each specific SSTR agonist was tested on Ca2+ currents to find the maximal effective concentration. Activation of SSTR2 with 10–7 and 10–8 M L-797,976 decreased the voltage-gated Ca2+ current and abolished any further decrease by SRIF. SSTR1, SSTR3, SSTR4, and SSTR5 agonists at 10–7 M did not modify the voltage-gated Ca2+ current and did not affect the Ca2+ current response to SRIF. These results indicate that SSTR2 is involved mainly in regulating voltage-gated Ca2+ currents by SRIF, which contributes to the decrease in intracellular Ca2+ concentration and GH secretion by SRIF.

somatostatin receptor subtypes; somatostatin receptor agonist; somatotrope

GROWTH HORMONE (GH) is secreted from the anterior pituitary and is regulated mainly by three hypothalamic hormones: GH-releasing hormone, ghrelin, and somatostatin (SRIF). SRIF is synthesized and released in the hypothalamus and transported to the anterior pituitary gland through the portal circulation. The physiological actions are initiated by the interaction between SRIF and specific, membrane-bound, high-affinity somatostatin receptors (SSTRs) on the surface of responsive cells. Five subtypes of SSTRs (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) have been identified as G protein-coupled seven-transmembrane domain receptors (28, 32). Activation of SSTRs is associated with a reduction in intracellular cAMP levels and intracellular Ca2+ concentration ([Ca2+]i) and stimulation of protein tyrosine phosphatase (30). SSTRs are coupled to several types of Ca2+ channels, the inhibition of which causes a decrease in Ca2+ currents leading to a decrease in the frequency and amplitude of action potentials, resulting in a reduction in [Ca2+]i (7). Nonpeptide agonists of each of the five SSTRs have been identified (SSTR1: L-797,591; SSTR2: L-779,976; SSTR3: L-796,778; SSTR4: L-803,087; SSTR5: L-817,818), and each agonist shows high affinity for its specific SSTR (35). Recently, Yang et al. (52) showed regulation of SSTRs by SRIF on K+ currents with the use of these five SSTR agonists.

Ion channels in pituitary cells are involved in the control of cell excitation that leads to hormone secretion. Although our understanding of hormone secretion resulting from generalized stimulation of SSTRs is quite advanced, specific roles for the different SSTR isoforms and the exact role of various ion channels in hormone secretion are not fully understood. Such an understating is required if the full therapeutic potential of selective SSTR agonists is to be achieved. Some progress has been made at the single cell level with patch-clamp techniques in conjunction with molecular biological approaches. Three cation channels, conducting Na+, K+, or Ca2+, have been identified in the membrane somatotropes, and these determine the electrical activity in these cells. Secretion of hormone from somatotropes is critically dependent on the level of free [Ca2+]i, which is controlled mainly by Ca2+ influx through voltage-gated Ca2+ channels and release from intracellular Ca2+ storage sites (7). In excitable cells, the voltage-gated Ca2+ channels play a key role in the control of GH secretion from the pituitary (7, 15, 21). Opening of voltage-gated Ca2+ channels in response to an appropriate change in membrane potential, which may also be modulated by second messenger systems, allows Ca2+ to move down the electrochemical gradient into the cytoplasm. The ensuing rise in [Ca2+]i leads to exocytosis of hormone or transmitter. Blockade of Ca2+ channels not only reduces basal GH secretion but also abolishes the inhibitory effect of SRIF on GH release (8). Therefore, Ca2+ influx through Ca2+ channels in the cell membrane is necessary for both exocytosis and inhibitory actions of factors such as SRIF. Five subtypes of voltage-gated Ca2+ channels (L, long-lasting; T, transient; N, neuronal; P/Q, Purkinje; R, residual) have been identified in somatotropes (19, 22, 33, 41) based on their electrophysiological and pharmacological properties (23, 26, 31, 42).

The inhibitory effect of SRIF involves an increase in K+ currents and a decrease in Ca2+ current so that the frequency and duration of action potentials are reduced, which subsequently leads to the reduction in Ca2+ influx and GH release (6–8). It was shown that SRIF increase the voltage-gated K+ current mainly through SSTR2 and SSTR4 (52). Although SSTRs have been linked to a reduction in GH release (27) or [Ca2+]i (3), the subtype(s) of SSTRs that mediates the decrease in voltage-gated Ca2+ currents by SRIF is still unknown. In the present study, we tested the effect of SSTR subtype-specific agonists on voltage-gated Ca2+ to clarify the subtypes of SSTRs involved in the decrease in the Ca2+ currents by SRIF in somatotropes.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Chemicals. Culture medium and FCS were purchased from Thermo Electron (Melbourne, Australia). Penicillin-streptomycin Fungizone antibiotic solution and trypsin were from Life Technologies (Gaithersburg, MD). Specific agonists of SSTR1–SSTR5 were provided by Merck Research Laboratories. SRIF was purchased from Auspep (Melbourne, Australia), and TTX, {omega}-conotoxin GVIA, and {omega}-agatoxin IVA were purchased from Alomone Laboratory (Jerusalem, Israel). Tetraethylammonium chloride, nifedipine, nystatin, DMSO, and all general salts for recording solutions were purchased from Sigma (St. Louis, MO).

Cell culture and preparation. The GH3 cells (American Type Culture Collection, Manassas, VA), originally obtained from the rat pituitary tumors, synthesize and release prolactin and GH. The GH3 cells were grown as monolayers in 80-cm2 plastic culture flasks (Nunc, Roskilde, Denmark) containing culture medium of 44.5% DMEM, 44.5% Ham's F-12, 10% FCS, and 1% penicillin-streptomycin (changed every 2 days) in a humidified atmosphere. Cells were harvested or passaged during the logarithmic phase of growth, at which time they were visibly confluent in the flask. Cells were plated onto 35-mm culture dishes for electrophysiological recording at the time of cell passage. Electrophysiological recordings were performed after 2–5 days in culture in 35-mm culture dishes.

Electrophysiological recording. On the day of recording, culture medium was replaced by patch-clamp bath solution at least 10 min before recording was started. Transmembrane Ca2+ currents were recorded by the patch-clamp technique in the nystatin-perforated, whole cell recording (WCR) configuration. Electrodes were pulled by a Sutter P-87 microelectrode puller (Sutter Equipment) from borosilicate micropipettes with inner filament (Harvard Apparatus, Edenbridge, UK). After fire polishing was completed, these electrodes had an initial input resistance of 3–5 M{Omega}. All recordings were made with the Axopatch-1C amplifier (Axon Instruments) or HEKA EPC 9 (HEKA Electronik, Wiesenstrasse, Germany).

The bath solution was composed of (in mM) 40 tetraethylammonium chloride, 90 NaCl, 5 CaCl2, 0.5 MgCl2, 10 glucose, and 10 HEPES (pH 7.4, adjusted with NaOH; osmolarity of 310 mosM). To exclude contamination by Na+ currents, 1 µM TTX was added to the bath solution during the experiment.

The pipette solution consisted of (in mM) 55 CsCl, 75 Cs2SO4, 8 MgSO4, and 10 HEPES (pH 7.4 and osmolarity of 310 mosM), and the electrodes were backfilled with this solution containing nystatin (300 µg/ml in 0.1% DMSO). This concentration of DMSO, applied to the cells studied in the WCR configuration, had no effect on membrane conductance.

Culture dishes containing the cells were fixed on the stage of an Olympus inverted microscope (New Hyde Park, NY). The electrode was positioned with a micromanipulator (Narishige). After a high-resistance seal was obtained, the pipette potential was held at –80 mV and voltage steps (10 mV, 250-ms duration) were delivered periodically to monitor the capacitance and access resistance. Access to the cell interior was judged by the appearance of a membrane capacitance transient current 2–5 min after the seal was formed. Whole cell capacitance and series resistance (only with cells with <35 M{Omega}) were compensated (>80%) before experimentation, and leak current was routinely subtracted with the Clampex 8.0 program (Axon Instruments) and Pulse program for EPC9 (HEKA Electronik). The change in series resistance over the course of each experiment was also monitored, and recordings with a significant change (>20%) in series resistance were excluded from the final data analysis. The electrical signal was filtered at 2 kHz with a low-pass filter, and the sweeps were sampled at 1 kHz in our recording protocols.

SRIF and some Ca2+ channel blockers (Ni+ and nifedipine, mixed with bath solution) were applied by a gravity pressure perfusion system at a rate of ~1 ml/min, and other Ca2+ channel blockers ({omega}-conotoxin GVIA and {omega}-agatoxin IVA) and each SSTR-specific agonist were added by hand to the culture dishes containing cells to be studied. Control recordings were started at least 10 min after the cell was patched. The effect of any drug treatment was recorded until the response reached a plateau or until we were certain that no change was occurring. The vehicle constituted the drug solvent and was applied at the same volume as used for drug application. Vehicle alone did not change the voltage-gated Ca2+ currents in our recording system. All experiments were performed at room temperature (20–22°C).

Varying the concentration of SSTR1–SSTR5 agonists. The specific agonists for SSTR1–SSTR5 used in this study are L-797,591 (SSTR1), L-779,976 (SSTR2), L-796,778 (SSTR3), L-803,087 (SSTR4), and L-817,818 (SSTR5). For SSTR2, 10–10 to 10–7 M was tested, and a concentration of 10–7 M was used for the other SSTRs. The final concentration of SRIF was 200 nM.

Data analysis. pCLAMP 8.0 software (Axon Instruments) and Pulse EPC 9 program (HEKA Electronik) were used to record and analyze the data. The effects of SSTR agonists were tested with ANOVA with post hoc testing and Student's paired t-test as appropriate to evaluate the statistical significance of differences between control and treated group means obtained from the same group of cells. Differences were considered to be significant at the level of P < 0.05. Group data are expressed as means ± SE. The example traces displayed were chosen as representative of at least four recordings under the same experimental conditions, unless otherwise indicated in the text.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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SRIF inhibits the voltage-gated Ca2+ current. In the presence of K+ and Na+ channel blockers, voltage-gated Ca2+ currents were recorded with the use of nystatin-perforated WCR configuration. Application of SRIF (200 nM) significantly reduced total voltage-gated Ca2+ currents evoked by depolarization steps from a holding potential of –80 mV (Fig. 1). From a holding potential of –80 mV, the maximal current was observed at a depolarizing step to 0 mV in these cells. Mean (±SE) values of maximal currents in response to step depolarization to 0 mV in six cells are shown in Fig. 1B. The reduction in the Ca2+ currents by SRIF was statistically significant (P < 0.05). The effect was reversible, with total recovery of peak current observed 10 min after the removal of SRIF (Fig. 1, A and B). The inhibitory effect of SRIF on total Ca2+ current is also demonstrated in the current-voltage relationship curve (Fig. 1C), which shows that no kinetic change occurred on application of SRIF. The effect of SRIF was reversible (Fig. 1C).


Figure 1
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  		Fig. 1. Effect of somatostatin (SRIF) on the voltage-gated Ca2+ current. Voltage-gated Ca2+ currents in GH3 cells were recorded from a holding potential of –80 mV. A: data from a representative cell during control conditions, SRIF (200 nM) treatment, and recovery. B: mean (±SE) Ca2+ current amplitude measured during depolarization steps to 0 mV (n = 6). *P < 0.05. C: current-voltage relationship of peak Ca2+ current amplitude with control, during application of SRIF, and after removal of SRIF (recovery).

 
Characterization of voltage-gated Ca2+ currents in GH3 cells. Five types of voltage-gated Ca2+ currents have been characterized in different cell types (49). In this study, we used specific Ca2+ channel blockers to characterize the Ca2+ currents. Ni+ (T-type blocker), nifedipine (L-type blocker), {omega}-conotoxin GVIA (N-type blocker), and {omega}-agatoxin IVA (P/Q-type blocker) were applied to cells during recording to block these Ca2+ channels. Although several studies used SNX-482 as the R-type Ca2+ current blocker, there have been reports of only partial blockade of R-type current and no change of Ca2+ influx in GH3 cells by SNX-482 at a very high dose (48, 49). Figure 2A shows a continued recording from one representative cell. The total Ca2+ currents shown in Fig. 2Aa were evoked by depolarizing steps from a holding potential of –80 mV in 10-mV increments from –50 to +40 mV. Nifedipine (10 µM) reduced the total Ca2+ current by ~30% (Fig. 2Ab), suggesting the presence of a significant contribution of L-type channels. The remaining Ca2+ current was further suppressed (by 20%) by {omega}-conotoxin GVIA (2.5 µM) in the presence of nifedipine, indicating the presence of N-type current (Fig. 2Ac). {omega}-Agatoxin IVA (200 nM), in the presence of nifedipine and {omega}-conotoxin GVIA, was without significant effect on the Ca2+ current (Fig. 2Ad). The remaining Ca2+ current was abolished by Ni+ (0.5 mM), reflecting the presence of T-type current (Fig. 2Ae). Although Ni+ is not a highly selective blocker of the T-type current, as it also reduces the R-type current (47), the results presented here most probably reflect the T-type current because GH3 cells lack the R-type current (24, 53). The current-voltage curves of the peak currents are summarized in Fig. 2B. Maximal current was observed at step depolarizations to 0 mV.


Figure 2
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  		Fig. 2. Characterization of voltage-gated Ca2+ current. A: voltage-gated Ca2+ currents in response to depolarizing steps from a holding potential of –80 mV in increments of 10 mV from –50 to +40 mV as shown in the pulse protocols (representative traces shown). Voltage-gated Ca2+ currents were evoked in control condition (a), in the presence of L-type blocker nifedipine (NFD; 10 µM) (b), in the presence of N-type blocker {omega}-conotoxin GVIA (2.5 mM) + NFD (c), in the presence of P/Q-type blocker {omega}-agatoxin IVA (200 nM) + NFD + {omega}-conotoxin GVIA (d), and in the presence of T-type blocker Ni+ (0.5 mM) + NFD, {omega}-conotoxin GVIA, and {omega}-agatoxin IVA (e). B: current-voltage relationship of Ca2+ current shown in A in control condition ({blacklozenge}), in the presence of NFD ({blacksquare}), in the presence of {omega}-conotoxin GVIA ({blacktriangleup}), in the presence of {omega}-agatoxin IVA ({lozenge}), and in the presence of Ni+ ({square}) measured at the peak of each current trace.

 
The next experiment was to determine which type or types of Ca2+ currents are involved in the action of SRIF in GH3 cells. To isolate T-, L-, and N-type currents, a combination of blockers was applied to the cells and SRIF (2x10–7M) was applied to determine its effect on each type of Ca2+ current. The peak Ca2+ currents in response to depolarizing steps from –80 to 0 mV are shown in Fig. 3. When the predominantly T-type current was isolated, in the presence of nifedipine, {omega}-conotoxin GVIA, and {omega}-agatoxin IVA, addition of SRIF had no additional effect (Fig. 3A). L-type current, isolated in the presence of Ni+, {omega}-conotoxin GVIA, and {omega}-agatoxin IVA, was significantly reduced by SRIF (Fig. 3B; P < 0.05). N-type current, isolated in nifedipine, Ni+, and {omega}-agatoxin IVA, was also significantly decreased by SRIF (Fig. 3C; P < 0.05).


Figure 3
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  		Fig. 3. L- and N-type Ca2+ current are decreased by SRIF in GH3 cells. Mean [±SE; n = 5 (A) and n = 4 (B and C)] Ca2+ current amplitudes measured during depolarizing steps to 0 mV from a holding potential of –80 mV are shown. A: lack of effect of SRIF on T-type Ca2+ current. B: effect of SRIF on L-type Ca2+ current. SRIF decreased L-type current significantly (*P < 0.05). C: effect of SRIF on N-type Ca2+ current. SRIF decreased N-type current significantly (*P < 0.05).

 
Effect of SSTR subtype-selective agonists on Ca2+ currents. The dose-response relationship of Ca2+ current to SSTR2 stimulation was constructed by stepping to 0 mV from a holding potential of –80 mV. The amplitude of the Ca2+ current was reduced by L-779,976 in a concentration-dependent manner (data from 6 cells, P < 0.05; Fig. 4). No difference was observed between 10–8 and 10–7 M L-779,976, indicating clearly that 10–8 M is the most effective concentration for this SSTR2 agonist. During application of 10–10, 10–9, 10–8, and 10–7 M SSTR2 agonist, a subsequent application of SRIF at the maximally effective dose of 10–8 M tested for further reduction in Ca2+ current in six cells (Fig. 5). L-779,976 (10–8 and 10–7 M) decreased the Ca2+ current significantly (P < 0.05) (as shown in our earlier experiments), and application of SRIF in the presence of SSTR2 agonist had no additional effect (Fig. 5, A and B). L-779,976 (10–9 M) reduced the Ca2+ current submaximally, and SRIF further reduced the Ca2+ current (P < 0.05; Fig. 5C). At 10–10 M, L-779,976 had no effect on the Ca2+ current, but these currents were significantly reduced by SRIF (Fig. 5D). These results indicate that SRIF reduces Ca2+ currents mainly through SSTR2.


Figure 4
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  		Fig. 4. Concentration response of SSTR2 agonist on voltage-gated Ca2+ current. Mean (±SE; n = 6) Ca2+ current amplitudes measured in response to depolarizing steps to 0 mV from a holding potential of –80 mV are presented. SSTR2 agonist decreased the Ca2+ current in a concentration-dependent manner with a maximum effect at 10–8 M. *P < 0.05.

 

Figure 5
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  		Fig. 5. Effect of SSTR2 agonist (10–10 to 10–7 M) on Ca2+ current amplitude and the additional response to SRIF. Mean (±SE; n = 6) Ca2+ current amplitudes in response to depolarizing steps to 0 mV from a holding potential of –80 mV are presented. SSTR2 agonist L-779,976 at 10–7 M (A) and 10–8 M (B) significantly (*P < 0.05) decreased the Ca2+ current, but additional SRIF had no effect. L-779,976 at 10–9 M (C) significantly (*P < 0.05) decreased the Ca2+ current, and a further reduction of the Ca2+ current occurred (*P < 0.05) on additional application of SRIF. D: 10–10 M did not alter Ca2+ current, but addition of SRIF significantly (*P < 0.05) decreased the Ca2+ current.

 
The maximal concentration of SSTR1, SSTR3, SSTR4, and SSTR5 subtype agonists (10–7 M) (52) was applied, with a subsequent application of SRIF (10–8 M), and the mean values of Ca2+ currents in six cells are shown in Fig. 6. Activation of SSTR1, SSTR3, SSTR4, and SSTR5 by their specific agonists did not modify the Ca2+ current, but SRIF application in the presence of SSTR1, SSTR3, SSTR4, and SSTR5 agonists decreased the Ca2+ current significantly (P < 0.05; Fig. 6), indicating that these receptor subtypes were not involved in the Ca2+ current response to SRIF.


Figure 6
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  		Fig. 6. Effect of SSTR1 (A), SSTR3 (B), SSTR4 (C), and SSTR5 (D) agonists (10–7 M) on the Ca2+ current and the additional response to SRIF. Mean (±SE; n = 6) Ca2+ current amplitudes measured in response to depolarizing steps to 0 mV from a holding potential of –80 mV are presented. SSTR1, SSTR3, SSTR4, and SSTR5 had no effect on the Ca2+ current, and application of additional SRIF decreased the Ca2+ current significantly (*P < 0.05).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Five subtypes of SSTRs have been cloned and identified as SSTR1–SSTR5 from various species (51). GH3 cells from a rat pituitary cell line, which are widely used in research, express and possess all five SSTRs (29). SRIF or SRIF agonists bind to SSTRs, activating G proteins and various downstream second messenger systems, resulting in suppression of voltage-gated Ca2+ currents and Ca2+ influx (5). In the present study, we examined which SSTR subtypes are involved in the reduction of voltage-gated Ca2+ currents by SRIF in GH3 cells using SSTR subtype agonists. Activation of SSTR1, SSTR3, SSTR4, and SSTR5 had no effect on either Ca2+ currents or the Ca2+-current response to SRIF. These four receptor subtypes are therefore not involved in SRIF-induced changes in Ca2+ current. Full activation of SSTR2 decreased the amplitude of Ca2+ currents to the same level as that induced by SRIF alone. Although a submaximal dose of SSTR2 agonist (10–9 M) evoked a significant decrease in the Ca2+ current, SRIF induced a further significant reduction, indicating that this dose of SSTR2 agonist only partially activated the signal transduction system. It is therefore suggested that stimulation of SSTR2 alone is the signaling pathway employed by SRIF to reduce voltage-gated Ca2+ currents in somatotropes. This correlates with the result from confocal fluorescence studies of [Ca2+]i, which is directly related to GH secretion, using a GC cell, which is derived from not somatotroph tumor cell (subclone of GH3 cell), in which the activation of SSTR2 almost completely blocked Ca2+ transients (3). Also, a previous report has shown a 40% inhibition of GH secretion in rat pituitary cells on stimulation of SSTR2 using the SSTR2 agonist L-779,976 (27), and that value was similar to the ~30% reduction in Ca2+ current observed in our study.

On the basis of kinetics, voltage dependency, and pharmacological sensitivities, five major types of voltage-gated Ca2+ currents have been identified: T, L, N, P/Q, and R type. In our experiments, based on pharmacological characteristics, the major contributors to the voltage-gated Ca2+ current are the T- and L-type Ca2+ channels, with a small involvement of N-type channels. There was no significant change in response to P/Q Ca2+ channel blockade. Previous studies on GH3 cells reported a small T-type current (~10% of total current) and a large L-type current component (2, 11). In the present study, we also observed a large contribution from T-type channels (~40% of total peak current) and L-type current similar to previous reports (2, 11). A concentration of 10 µM nifedipine, which is sufficient to inhibit more than ~90–95% of the L-type Ca2+ current but is low enough not to block other subtypes of Ca2+ channels (20), was used to test the involvement of L-type Ca2+ current. No known pharmacological agent specifically targets T-type Ca2+ channels. Ni+ is somewhat more specific for T-type Ca2+ current (17), and in high concentrations it may block other types of Ca2+ channels; thus 0.5 mM Ni+ is generally used. Thus, in our experiments, Ni+ was used last to block Ca2+ current resistant to nifedipine, conotoxin, and agatoxin. It is therefore concluded that the current sensitive to Ni+ may be through T-type channels. Mibefradil and amiloride are T-type channel antagonists (1, 18, 37, 46), which, among three subunits of T-type channels, antagonize two subunits. Ni+ binds to all three subunits (10); therefore, mibefradil and amiloride are less potent than Ni+. N-type Ca2+ current has not been reported extensively in pituitary cells, but a study characterized 20% of total Ca2+ currents as {omega}-conotoxin GVIA-sensitive N-type currents in GH3 cells (56). Herein, ~15% of the Ca2+ current was blocked by {omega}-conotoxin GVIA in GH3 cells. Previous studies have shown that, at concentrations >100 nM, {omega}-agatoxin IVA can block P/Q-type Ca2+ currents (23, 26). In our experiments, {omega}-agatoxin IVA (200 nM) was without significant effect on the Ca2+ current. P/Q-type channels are thus not involved in the total Ca2+ current in GH3 cells. Our experiments showed that the majority of the voltage-gated Ca2+ current was composed of T-, L-, and N-type currents, with a large proportion of T- and L-type and a smaller proportion of N-type currents. Only L- and N-type Ca2+ currents were reduced by SRIF in GH3 cells.

From our dose-response data for SSTR2 agonist, 10–8 M is the lowest dose giving a maximal response, which coincides with previous receptor-binding results (36, 43, 44) and K+ current responses published previously (52). We observed less reduction at a higher dose (10–7 M), which may reflect desensitization of SSTRs, and we observed a similar effect on K+ current responses in these cells (52). Several studies have shown that the SRIF ligand-receptor complex undergoes internalization and desensitization (14, 20, 38, 50) through uncoupling of the activated complex from G protein by receptor phosphorylation, a common regulatory feature of many G-protein-coupled receptors (25). SSTR2 seems to undergo rapid phosphorylation, leading to rapid desensitization of receptors (13, 39). In neurons, desensitization of SSTRs occurs in a concentration- and exposure time-dependent manner. [Ca2+]i spikes were reduced by SRIF but returned to control levels several minutes after application of a higher dose of SRIF (0.1–1 µM) (54). Among the five receptors, only receptor 2 is involved in Ca2+ current modification. It has been found that the five receptors play different roles in different systems and that they function by different mechanisms. SSTR2 is also involved in K+ current modification (52) and in inhibition of cell growth and induction of apoptosis (12, 16, 34). SSTR1 is involved in cell growth regulation (55); SSTR3 mediates apoptosis (40); SSTR4 is involved in K+ current modification (52), but other functions remain unknown. SSTR5 mediates growth inhibition and cell proliferation and has been shown to be important in cancer growth regulation as one of the most potent inhibitory receptors (9, 45).

The results of our study demonstrate that the voltage-gated Ca2+ current in GH3 cells is significantly and reversibly decreased by SRIF. This result is in agreement with several reports that SRIF decreases Ca2+ current in primary cultured rat and ovine somatotropes (5, 6, 8). This would lead to a reduction in the level of [Ca2+]i via a decrease Ca2+ influx and reduction in GH secretion. It was previously shown that voltage-gated K+ current was increased by SSTR2 and SSTR4, leading to hyperpolarization of the cell membrane and decreased GH secretion (52). Therefore, SSTR2 and SSTR4 are the main receptors to activate voltage-gated K+ current, and SSTR2 is the main receptor inhibiting voltage-gated Ca2+ current. SSTRs are G protein-coupled receptors, for which Gi protein mediates the effect on voltage-gated K+ channels and Go protein mediates the effect on voltage-gated Ca2+ channels (4, 5). Because SSTRs influence Ca2+ and K+ currents by different G proteins, SSTR2 may be coupled to two different G proteins to mediate the effect on voltage-gated Ca2+ and K+ currents, respectively.

In conclusion, using selective SSTR agonists and ion-channel blockers, we have demonstrated that SSTR2 is involved in the SRIF-induced decrease in voltage-gated Ca2+ currents, where L- and N-type Ca2+ currents are reduced by SRIF in GH3 cells. The reduction in Ca2+ currents by SRIF would decrease Ca2+ influx and the level of [Ca2+]i in GH3 cells, leading to a decline in GH secretion. Because SSTR2 mediates the effect of SRIF on both Ca2+ and K+ currents via Go and Gi proteins, it is suggested that one G protein-coupled receptor may be linked to two different G proteins.


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This work is supported by funding from National Health and Medical Research Council of Australia to C. Chen.


   ACKNOWLEDGMENTS
 
We thank Dr. A. D. Black (Seton Hall University, South Orange, NJ) and Merck Research Laboratories for providing the SRIF agonists. We thank Dr. D. J. Keating for comments and technical help in early stages of this work and Drs. Y. F. Zhao and E. Vargas for scientific contributions during discussion. We are also indebted to M. Hernandez and K. Wang for help on cell preparation and D. Arnold for manuscript editing.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Chen, Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia (e-mail: Chen.Chen{at}princehenrys.org)

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. Section 1734 solely to indicate this fact.


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 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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