Somatostatin Increases Voltage-Gated K+ Currents in GH3 Cells through Activation of Multiple Somatostatin Receptors
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《内分泌学杂志》
Prince Henry’s Institute of Medical Research (S.-K.Y., D.J.K., C.C.) and Department of Physiology (S.-K.Y., H.C.P., C.C.), Monash University, Melbourne 3168, Australia
Department of Biology (A.D.B.), Seton Hall University, South Orange, New Jersey 07079
Abstract
The secretion of GH by somatotropes is inhibited by somatostatin (SRIF) through five specific membrane receptors (SSTRs). SRIF increases both transient outward (IA) and delayed rectifying (IK) K+ currents. We aim to clarify the subtype(s) of SSTRs involved in K+ current enhancement in GH3 somatotrope cells using specific SSTR subtype agonists. Expression of all five SSTRs was confirmed in GH3 cells by RT-PCR. Nystatin-perforated patch clamp was used to record voltage-gated K+ currents. We first established the presence of IA and IK type K+ currents in GH3 cells using different holding potentials (–40 or –70 mV) and specific blockers (4-aminopirimidine and tetraethylammonium chloride). SRIF (200 nM) increased the amplitude of both IA and IK in a fully reversible manner. Various concentrations of each specific SRTR agonist were tested on K+ currents to find the maximal effective concentration. Activation of SSTR2 and SSTR4 by their respective agonists, L-779,976 and L-803,087 (10 nM), increased K+ current amplitude without preference to IA or IK, and abolished any further increase by SRIF. Activation of SSTR1 and SSTR5 by their respective agonists, L-797,591 or L-817,818 (10 nM), increased K+ current amplitude, but SRIF evoked a further increase. The SSTR3 agonist L-797,778 (10 nM) did not affect the K+ currents or the response to SRIF. These results indicate that SSTR1, -2, -4, and -5 may all be involved in the enhancement of K+ currents by SRIF but that only the activation of SSTR2 or -4 results in the full activation of K+ current caused by SRIF.
Introduction
GH IS AN ANABOLIC hormone that is synthesized, stored, and secreted by pituitary somatotrope cells. GH secretion is primarily under the reciprocal control of two hypothalamic hormones, a stimulatory GHRH and an inhibitory hormone, somatostatin (SRIF). SRIF is synthesized in the hypothalamus and released into and transported by the hypothalamo-hypophyseal portal blood vessels to the anterior pituitary gland. The physiological actions of SRIF are initiated by its interaction with specific plasma membrane-bound SRIF receptors (SSTRs). Five different SSTR subtypes, SSTR1, -2, -3, -4, and -5, have been cloned and characterized (1). Each of these receptors contains seven a helical transmembrane segments, which is typical for G protein-coupled receptors (2). Ligand activation of SSTRs is associated with a reduction in intracellular cAMP levels and Ca2+ concentration and stimulation of protein tyrosine phosphatase (3). SSTRs are coupled to several types of K+ channels whose activation causes hyperpolarization of the membrane, leading to the cessation of spontaneous action potential activity facilitating a reduction in intracellular Ca2+ concentration (3, 4).
Nonpeptide agonists of each of the five SSTRs, L-797,591 (SSTR1), L-779,976 (SSTR2), L-796,778 (SSTR3), L-803,087 (SSTR4), and L-817,818 (SSTR5), have been created by the Merck Research Laboratories (South Orange, NJ) (5). Each agonist shows high affinity for their specific SSTR subtype. For example, L-797,591 shows 100 times higher affinity for SSTR1, compared with SSTR4, whereas L-817,818 has an affinity 130 times higher for SSTR5 than SSTR2 and 8 times higher than SSTR1 (5). These agonists have previously been used to study the physiological function of SSTR subtypes in regulating hormone secretion (6, 7).
Ion channels in most pituitary cells are involved in the control of cell excitation, which regulates hormone secretion. Na+, K+, and Ca2+ channels have been identified in the plasma membrane of somatotropes and determine their electrical activity. Hormone secretion from somatotropes is triggered by an increase in intracellular-free Ca2+ ([Ca2+]i) caused by Ca2+ influx through voltage-gated Ca2+ channels (8). Na+ and K+ channels are involved in the modification of somatotrope function via their effect on membrane potential and action potential duration and hence [Ca2+]i (8). It is well documented that a number of neuropeptides, especially those from the hypothalamus, exert their regulatory role on somatotrope hormone secretion through the modification of transmembrane ion channels (8, 9, 10).
In excitable cells, most outward current is carried by K+ ions (11). In somatotropes, at least three types of voltage-gated K+ current have been characterized according to their pharmacology, voltage, and Ca2+ dependency and kinetic properties, namely transient outward (IA), delayed rectifying (IK) and inward rectifying (Kir) currents (12, 13, 14). IA is evoked at holding potentials more negative than –70 mV, is closed at potentials above –40 mV, and displays rapid inactivation. The IA current in rat somatotropes is kinetically similar to that seen in bovine somatotropes, rat lactotropes, and the GH3 somatotrope cell line (10, 15, 16) The IA channel antagonist 4-aminopirimidine (4-AP) stimulates GH secretion in ovine somatotropes, indicating that IA channels may be essential for maintaining the resting membrane potential and determining [Ca2+]i (13). IK is activated at around –20 mV from a holding potential of –40 mV and is slowly inactivating (13). Kir channels play an important role in maintaining resting membrane potential (14) but contribute only a small portion of total K+ currents in somatotropes (17).
The inhibitory effect of SRIF could be explained, at least partially, by the fact that SRIF increases K+ currents so that the frequency and duration of action potentials are reduced, which subsequently leads to the reduction in Ca2+ influx and GH release (4, 8, 12). In the present study, we tested the effect of SSTR subtype-specific agonists on voltage-gated K+ currents to clarify the subtype(s) of SSTRs involved in the increase in the K+ currents by SRIF in somatotropes.
Materials and Methods
Chemicals
Culture medium and fetal calf serum were purchased from Thermo Electron Corp. (Melbourne, Australia). Penicillin-streptomycin Fungizone antibiotic solution and trypsin were from Life Technologies, Inc. (Gaithersburg, MD). SSTR1–5-specific agonists were provided by Merck Research Laboratories. SRIF was purchased from Auspep (Melbourne, Australia), and tetrodotoxin, charybdotoxin (ChTX), and apamin were purchased from Alomone Laboratory (Jerusalem, Israel). Tetraethylammonium chloride (TEA), 4-AP, nifedipine, nystatin, dimethyl sulfoxide (DMSO), and all general salts for recording solutions were purchased from Sigma (St. Louis, MO).
Multiplex RT-PCR of SRIF receptor subtypes
PCR primer sequences for each of the rat SSTR subtypes were as follows: SSTR1, sense, 5'-CTA CTT TGC CGC CTG GTG CTC-3', and antisense, 5'-TGG CAA TGA TGA GCA CGT AAC-3' [GenBank accession no. (ACC) X62314]; SSTR2, sense, 5'-TTG ACG GTC ATG AGC ATC G-3', and antisense, 5'-ACA GAC ACG GAC GAG ACA TTG-3' (ACC M93273); SSTR3, sense, 5'-GGC CGC TGT TAC CTA TCC TTC-3', and antisense, 5'-GGC ACT CCT GAG AAC ACA ACC-3' (ACC X63574); SSTR4, sense, 5'-CGG AGA CGC TCA GAG AAG AAG-3', and antisense, 5'-TGG TCT TGG TGA AAG GGA CTC-3' (ACC M96544); SSTR5, sense, 5'-CAT GAG TGT TGA CCG CTA CC-3', and antisense, 5'-GGC ACA GCT ATT GGC ATA AG-3' (ACC L04535). The expected sizes of PCR products were 364 bp for SSTR1, 449 bp for SSTR2, 555 bp for SSTR3, 409 bp for SSTR4, and 508 bp for SSTR5. The SSTR subtype mRNA levels were obtained by RT-PCR, and all the procedures were taken from Park et al. (18). We have confirmed expression of SSTR1–5 in GH3 cells by RT-PCR. All PCRs were performed in a 25-μl volume containing 2 μl reverse transcription reaction, 2x Master Mix buffer (consisting of 3 mM MgCl2; 400 μM deoxyATP, deoxyGTP, deoxyCTP, deoxythymidine triphosphate; and 50 U/ml Taq DNA polymerase) (Promega Corp., Madison, WI). The thermal cycling profile was as follows: 95 C for 10 min, 24 (SSTR2, SSTR5) or 28 (SSTR1, SSTR3, SSTR4) cycles of 95 C for 30 sec, 60 C for 1 min, and 72 C for 1 min. The final extraction was at 72 C for 10 min. Reaction products were separated on a 1.4% gel and analyzed using the Quantity One gel program (Gel-DOC 2000; BioRad, Segrate, Italy).
Cell culture and preparation
The GH3 cell line (American Type Culture Collection, Manassas, VA), originally obtained from the rat pituitary, secretes prolactin and GH and secretion is regulated by GHRH and SRIF. The GH3 cells were grown as monolayers in 80-cm2 plastic culture flasks (Nunc Brand Product, Roskilde, Denmark) containing culture medium in a humidified atmosphere containing 5% CO2 at 37 C. The culture medium, which is composed of 44.5% DMEM, 44.5% Hams F-12, 10% fetal calf serum, and 1% penicillin streptomycin, was changed every 2 d. 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 d 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. Transmembrane currents were recorded using the patch-clamp technique in nystatin-perforated, whole-cell recording (WCR) configuration. Electrodes were pulled by a Sutter P-87 microelectrode puller (Sutter Equipment Co., Navato, CA) from borosilicate micropipettes with inner filament (Harvard Apparatus Ltd., Edenbridge, UK). After fire polishing, these electrodes had an initial input resistance of 3–5 M. All recordings were made using the Axopatch-1 C amplifier (Axon Instrument, Foster City, CA).
The bath solution contained 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4, adjusted with NaOH; osmolarity of 310 mOsm/liter). To exclude the occurrence of Ca2+ and Na+ currents, cobalt (Co2+, 3 mM), and tetrodotoxin (1 μM) were added to the bath solution on the day of experimentation.
The pipette solution contained 55 mM KCl, 75 mM K2SO4, 8 mM MgSO4, and 10 mM HEPES (pH 7.4 and osmolarity of 310 mOsm/liter), and the electrode was backfilled with this solution containing nystatin (300 μg/ml in 0.1% DMSO). This concentration of DMSO, applied to the cell studied in the WCR configuration, had no effect on membrane conductance.
Cell culture dishes were fixed on the stage of an inverted microscope (Olympus, New Hyde Park, NY). After obtaining a high-resistance seal using a micromanipulator (Narishige, Tokyo, Japan), the pipette potential was held at –70 mV and voltage pulses (10 mV, 200-msec 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 forming a seal. Whole-cell capacitance and series resistance (using only cells with <35 M) were compensated (>80%) before experimentation, and leak current was routinely subtracted using the Clampex 8.0 program (Axon Instruments). The change in series resistance over the course of each experiment was also monitored, and recordings with significant change (more than 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.
Each SSTR-specific agonist, SRIF, and K+ channel blockers (ChTX, apamin, 4-AP, TEA) were applied by a gravity pressure perfusion system at a rate of approximately 1 ml/min. Control recordings were started at least 10 min after patching the cell. 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 K+ currents in our recording system. All experiments were performed at room temperature (20–22 C).
Varying the concentration of SSTR 1–5
The specific agonists for SSTR1–5 used in this study were: L-797,591 (SSTR1), L-779,976 (SSTR2), L-796,778 (SSTR3), L-803,087 (SSTR4), and L-817,818 (SSTR5). To find the maximal response concentration, a range of doses were tested. For SSTR1 and SSTR5, 10–9, 10–8, and 10–7 M were tested, and for SSTR2 and SSTR4, 10–10, 10–9, 10–8, and 10–7 M were tested. For SSTR3, only 10–8 and 10–7 M were tested. The final concentration of each agonist that we used as maximal was 10–8 M and submaximal as 10–9 M. The final concentration of SRIF was 200 nM.
Data analysis
The pCLAMP 8.0 software (Axon Instruments) was used to record and analyze the data. Effect of SSTR agonists was tested using 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 mean ± SEM. The example traces displayed were chosen as representative of at least six recordings under the same experimental conditions unless otherwise indicated in the text.
Results
Multiplex RT-PCR for pituitary SSTR subtypes
We confirmed expression of SSTR1–5 in GH3 cells by multiplex RT-PCR (Fig. 1). The multiplex RT-PCR assay in this study amplified SSTR2 and SSTR5 or SSTR1, SSTR3, and SSTR4 transcripts from GH3 cell culture in a single tube, resulting in PCR products of the expected sizes (Fig. 1, A and B).
Characterization of the voltage-gated K+ current in GH3 cells
In the presence of blockers of Ca2+ and Na+ channels as detailed in Materials and Methods, voltage-gated K+ currents were recorded using nystatin-perforated WCR. To test for the presence of Ca2+-activated K+ currents (KCa), ChTX, a blocker of large conductance KCa channels, and apamin, a specific blocker of small conductance KCa channels, were applied to cells. Local application of ChTX (1 μM) or apamin (1 μM) did not modify the recorded K+ currents (results not shown). Addition of SRIF in the presence of ChTX and apamin increased voltage-gated K+ current amplitude significantly (results not shown) to the same level as SRIF alone, thus excluding the involvement of KCa currents in our experiments. K+ currents were evoked by voltage steps from a holding potential of either –70 mV (total currents) or –40 mV (IK currents only) to potentials of –50 to +60 mV, in increments of 10 mV over 200 msec (Fig. 2). The GH3 cells possessed both IA and IK currents. Only the IK current was recorded during depolarizations from a holding potential of –40 mV (Fig. 2Ab), whereas the IA (Fig. 2Ac) was revealed when this IK current was subtracted from total currents recorded with a holding potential of –70 mV (Fig. 2Aa). The current-voltage relationship for total K+ currents (IA and IK, Fig. 2Aa), the IK current (Fig. 2Ab), and the IA current (Fig. 2Ac) are shown in Fig. 2B. It is obvious that the IA current is a small component of total K+ currents in GH3 cells, compared with a large component attributable to the IK current.
In addition to different voltage-dependent characters, IA and IK channels have different sensitivities to some K+ channel antagonists. The IA channel is highly sensitive to 4-AP, whereas the IK channel can be blocked by TEA (12). As shown in Fig. 3, total K+ current was recorded from cells at a holding potential of –70 mV (Fig. 3Aa), whereas the transient component was absent in the presence of 4-AP (3 mM) (Fig. 3Ab). This result indicates an inhibition of predominantly the IA current. The current that persisted in the presence of 4-AP was predominantly the IK current, which was completely blocked by TEA (10 mM) (Fig. 3Ad). The current-voltage relationships of total peak current (IA and IK, Fig. 3Aa) and IK currents (Fig. 3Ab) are shown in Fig. 3B and are similar to the data obtained with different holding potentials in Fig. 2. The difference between these two traces (Fig. 3Ac) reveals the total 4-AP-sensitive IA current and is similar to the IA current revealed by the difference in currents obtained at holding potentials of –70 vs. –40 mV (Fig. 2Ac).
Time response of SRIF effects
To investigate the time course of the K+ current response to SRIF, total voltage-gated K+ currents were recorded with single steps from a holding potential of –70 to +60 mV every minute. The SRIF-induced increase in K+ currents occurred 2–3 min after application and reached a maximum after 10–12 min and recovered completely 10–15 min after removal of SRIF (Fig. 4).
SRIF activates the voltage-gated K+ current
Application of SRIF (200 nM) significantly increased the total voltage-gated K+ currents evoked by depolarizing steps from a holding potential of –70 mV (Fig. 5). SRIF application increased both IA and IK currents without preference because both the peak (IA and IK) and sustained (mostly IK) components of the response were enhanced equivalently (in Fig. 5, compare A1 and A2). In addition, current-voltage relationships of K+ currents before and after application of SRIF indicate that there is no kinetic change with the application of SRIF, although the amplitude of total currents at many depolarizing steps is increased (Fig. 5C). The effect was reversible, with total recovery of peak current observed 10 min after the removal of SRIF (Fig. 5A3). Mean (±SEM) values of current measured at a step to +60 mV in eight cells are shown in Fig. 5B and increase in the K+ currents by SRIF was statistically significant (P < 0.01). Both IA and IK currents were increased by SRIF (Fig. 6, A and B). The mean (±SEM) increase in IA and IK current amplitude at +60 mV by SRIF measured from four cells was statistically significant (Fig. 6C; P < 0.05).
Effect of different doses of SSTR agonists on K+ current
The dose-dependent increase in K+ current by SSTR agonists was carried out from a holding potential of –70 mV. Mean amplitude of the K+ current at +60 mV for each agonist is shown in Fig. 7. SSTR1 and SSTR5 agonists had no effect at 10–9 M, whereas 10–8 and 10–7 M significantly increased [P < 0.05 (SSTR1), P < 0.01 (SSTR5)] K+ current amplitude (Fig. 7, A and E). SSTR2 and SSTR4 agonists at 10–10 M did not affect K+ current amplitude, but exposure to 10–9 and 10–8 M increased it significantly (P < 0.01) over control, whereas 10–7 M of these agonists had no further effect. The SSTR3 agonist, at 10–8 or 10–7 M, did not affect K+ current amplitude, compared with control (Fig. 7C). It is therefore clear that 10–8 M is the maximal effective concentration for each agonist, apart from the SSTR3 agonist, whereas10–9 M activates the voltage-gated K+ currents to submaximal levels.
Effect of SSTR subtype-selective agonists on K+ current
The maximal concentration of each SSTR subtype agonist (10–8 M) was applied, with a subsequent application of SRIF and the effects on total K+ current from eight cells is shown in Fig. 8. SSTR1 and SSTR5 agonists increased K+ current significantly (P < 0.01), and addition of SRIF in the presence of these agonists further increased the K+ current significantly (Fig. 8, A and E). SSTR2 and SSTR4 agonists increased K+ current significantly (P < 0.01), but further addition of SRIF in the presence of these agonists had no additional effect on K+ current (Fig. 8, B and D). The SSTR3 agonist itself had no effect on the K+ current, but SRIF application in the presence of SSTR3 agonist increased the K+ current significantly (P < 0.01, Fig. 8C).
Because the SSTR1 agonist, L-797,591, can activate SSTR4 receptors, albeit at 100 times lower affinity than for SSTR1, and the SSTR5 agonist, L-817,818, showed 8 times higher affinity for SSTR5, compared with SSTR1, and 130 times higher than SSTR2 (5), the partial increase in the K+ current by L-797,591 and L-817,818 may reflect a cross-reaction of other SSTRs subtypes rather than on SSTR1 and SSTR5 specifically. Submaximal concentration, 10–9 M, of agonists was therefore tested followed by exposure to SRIF. SSTR2 and SSTR4 agonists at 10–9 M increased the K+ current significantly (P < 0.01), and addition of SRIF further increased the K+ current significantly (P < 0.05 and P < 0.01, Fig. 9, B and C). Although SSTR1 and SSTR5 agonists 10–9 M had no effect on the K+ current, addition of SRIF in the presence of these agonists increased the K+ current fully (P < 0.01, Fig. 9, A and D).
Discussion
Five subtypes of SSTRs have been cloned and identified from various species including human, rat, mouse, porcine, and bovine (19, 20). Binding of SRIF or SRIF ligands to SSTRs induces G protein activation and signaling through various pathways, resulting in activation of K+ channels and consequent suppression of voltage-gated Ca2+ influx (21, 22).
In this present study, we examined which subtype or subtypes of SSTR are involved in the modification of the voltage-gated K+ current by SRIF in GH3 cells using SSTR subtype agonists. Activation of SSTR3 has no effect on K+ current, ruling out its involvement in the SRIF-induced responses. Full activation of SSTR2 or SSTR4 increased the amplitude of the K+ current to the same level as that of SRIF alone. In contrast, SSTR1 and SSTR5 agonists produce only a partial increase in the K+ current. This partial activation of K+ current by the maximal concentration of SSTR1 and SSTR5 agonists may not actually be due to the activation of SSTR1 and -5 because these agonists have a moderate affinity for either SSTR2 or SSTR4 as well (5). A submaximal dose of agonists for SSTR1 and SSTR5 induced no effect on the K+ current, whereas the same dose of agonists for SSTR2 and SSTR4 evoked significant increase in the same K+ current. It is therefore possible that SSTR1 and SSTR5 may not be involved in the SRIF-induced increase in voltage-gated K+ currents due to the potential nonspecific effects of SSTR1 and -5 agonists on SSTR2 and -4. Whereas we cannot be completely certain of this conclusion from our current experiments, we can be certain that stimulation of either SSTR2 or -4 can fully activate K+ currents with no further increase by SRIF.
One possible explanation for this is that activation of either SSTR2 or SSTR4 alone is capable of fully activating the signal transduction system, which is responsible for the increase in voltage-gated K+ currents by SRIF. Thus, as long as one receptor type, either SSTR2 or SSTR4, is fully activated, maximal SRIF-induced increment of the K+ current can be achieved. Another possibility is that SSTR2 and SSTR4 undergo heterodimerization in GH3 cells. Recent studies have shown that SSTRs exhibit SRIF-induced homo- and heterodimerization, which changes their binding and functional properties (23, 24, 25). For example, heterodimerization of SSTR2 and SSTR3 in human embryonic kidney cells resulted in high-affinity binding to SRIF and SSTR2 agonist (L-779,976) but not to the SSTR3 agonist (L-797,778) (26). SSTR2 is most abundantly expressed in neural and neuroendocrine tissues (27), especially in the rat (28). In the rat pituitary, the rank of order of expression of SSTRs is SSTR2 > SSTR1 = SSTR3 > SSTR5 > SSTR4 (27, 29, 30). Although SSTR4 is expressed at high levels in the habenula and heart of rat (30, 31, 32), it is the least expressed SSTR in the pituitary of that species. Thus, activation of either SSTR2 or SSTR4 may induce dimerization, and this dimer then activates the voltage-gated K+ channels to increase K+ currents. The role of SSTR heterodimerization, if true, will have a major impact on future drug design (33).
Based on kinetics, voltage dependency, and pharmacological sensitivities, four major types of voltage-gated K+ currents identified in mammalian pituitary cells are Kir, IA, IK, and M-type K+ currents (10, 12, 13, 21). In different species there may be different proportions of each type of voltage-gated K+ channel. For example, IA makes up a large proportion of the total K+ current in rat pituitary cells but accounts for only a small component of the total K+ current in sheep somatotropes (12, 34). From our experiments, the majority of voltage-gated K+ currents were composed of IK currents, with a small proportion being IA current. We have demonstrated these two types of voltage-gated K+ currents based on their voltage-dependent kinetics and pharmacological characteristics.
Previous work on GH3 cells indicated low levels of two types of Ca2+-activated K+ currents (35). In the present study, Ca2+-activated K+ currents in GH3 cells seem to be absent under the recording conditions used in the presence of the Ca2+ channel blocker, Co2+ (36). The K+ current response to SRIF reported here does not involve Ca2+-activated K+ currents.
The results from our study demonstrate that voltage-gated K+ currents in GH3 cells are significantly and reversibly increased by application of SRIF. This result is in agreement with several reports that SRIF increases K+ current in primary cultured rat and ovine somatotropes (12, 13, 34). The functional consequence of this increase in K+ current would be to hyperpolarize the plasma membrane (10, 13, 37). This would reduce the probability of opening of voltage-gated Ca2+ channels and hence reduce the action potential duration and hence reduce [Ca2+]i and GH secretion.
In conclusion, using selective SSTR2 and -4 agonists, we have demonstrated that SSTR2 and SSTR4 are involved in the SRIF-induced increase in voltage-gated K+ currents in GH3 cells. This may be possible if stimulation of either SSTR2 or SSTR4 alone can fully activate the signal transduction system(s) responsible for the increase in K+ currents by SRIF. Alternatively, SSTR2 and SSTR4 may undergo heterodimerization; however, both these possibilities require further investigation. Activation of the voltage-gated K+ current by SRIF is likely to decrease the level of [Ca2+]i and GH secretion as a result of membrane hyperpolarization and a decrease in action potential frequency and/or duration.
Acknowledgments
We thank Ms. M. Hernandez and D. Feng for their help on cell preparation, Ms. D. Arnold for manuscript editing, and Ms. S. Panckridge for graphical preparation.
Footnotes
This work was supported by Australian National Health and Medical Research Council.
Abbreviations: 4-AP, 4-Aminopirimidine; [Ca2+]i, intracellular-free Ca2+; ChTX, charybdotoxin; DMSO, dimethyl sulfoxide; IA, transient outward K+ current; IK, delayed rectifying K+ current; KCa, Ca2+-activated K+ current; Kir, inward rectifying current; SRIF, somatostatin; SSTR, SRIF receptor; TEA, tetraethylammonium chloride; WCR, whole-cell recording.
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Department of Biology (A.D.B.), Seton Hall University, South Orange, New Jersey 07079
Abstract
The secretion of GH by somatotropes is inhibited by somatostatin (SRIF) through five specific membrane receptors (SSTRs). SRIF increases both transient outward (IA) and delayed rectifying (IK) K+ currents. We aim to clarify the subtype(s) of SSTRs involved in K+ current enhancement in GH3 somatotrope cells using specific SSTR subtype agonists. Expression of all five SSTRs was confirmed in GH3 cells by RT-PCR. Nystatin-perforated patch clamp was used to record voltage-gated K+ currents. We first established the presence of IA and IK type K+ currents in GH3 cells using different holding potentials (–40 or –70 mV) and specific blockers (4-aminopirimidine and tetraethylammonium chloride). SRIF (200 nM) increased the amplitude of both IA and IK in a fully reversible manner. Various concentrations of each specific SRTR agonist were tested on K+ currents to find the maximal effective concentration. Activation of SSTR2 and SSTR4 by their respective agonists, L-779,976 and L-803,087 (10 nM), increased K+ current amplitude without preference to IA or IK, and abolished any further increase by SRIF. Activation of SSTR1 and SSTR5 by their respective agonists, L-797,591 or L-817,818 (10 nM), increased K+ current amplitude, but SRIF evoked a further increase. The SSTR3 agonist L-797,778 (10 nM) did not affect the K+ currents or the response to SRIF. These results indicate that SSTR1, -2, -4, and -5 may all be involved in the enhancement of K+ currents by SRIF but that only the activation of SSTR2 or -4 results in the full activation of K+ current caused by SRIF.
Introduction
GH IS AN ANABOLIC hormone that is synthesized, stored, and secreted by pituitary somatotrope cells. GH secretion is primarily under the reciprocal control of two hypothalamic hormones, a stimulatory GHRH and an inhibitory hormone, somatostatin (SRIF). SRIF is synthesized in the hypothalamus and released into and transported by the hypothalamo-hypophyseal portal blood vessels to the anterior pituitary gland. The physiological actions of SRIF are initiated by its interaction with specific plasma membrane-bound SRIF receptors (SSTRs). Five different SSTR subtypes, SSTR1, -2, -3, -4, and -5, have been cloned and characterized (1). Each of these receptors contains seven a helical transmembrane segments, which is typical for G protein-coupled receptors (2). Ligand activation of SSTRs is associated with a reduction in intracellular cAMP levels and Ca2+ concentration and stimulation of protein tyrosine phosphatase (3). SSTRs are coupled to several types of K+ channels whose activation causes hyperpolarization of the membrane, leading to the cessation of spontaneous action potential activity facilitating a reduction in intracellular Ca2+ concentration (3, 4).
Nonpeptide agonists of each of the five SSTRs, L-797,591 (SSTR1), L-779,976 (SSTR2), L-796,778 (SSTR3), L-803,087 (SSTR4), and L-817,818 (SSTR5), have been created by the Merck Research Laboratories (South Orange, NJ) (5). Each agonist shows high affinity for their specific SSTR subtype. For example, L-797,591 shows 100 times higher affinity for SSTR1, compared with SSTR4, whereas L-817,818 has an affinity 130 times higher for SSTR5 than SSTR2 and 8 times higher than SSTR1 (5). These agonists have previously been used to study the physiological function of SSTR subtypes in regulating hormone secretion (6, 7).
Ion channels in most pituitary cells are involved in the control of cell excitation, which regulates hormone secretion. Na+, K+, and Ca2+ channels have been identified in the plasma membrane of somatotropes and determine their electrical activity. Hormone secretion from somatotropes is triggered by an increase in intracellular-free Ca2+ ([Ca2+]i) caused by Ca2+ influx through voltage-gated Ca2+ channels (8). Na+ and K+ channels are involved in the modification of somatotrope function via their effect on membrane potential and action potential duration and hence [Ca2+]i (8). It is well documented that a number of neuropeptides, especially those from the hypothalamus, exert their regulatory role on somatotrope hormone secretion through the modification of transmembrane ion channels (8, 9, 10).
In excitable cells, most outward current is carried by K+ ions (11). In somatotropes, at least three types of voltage-gated K+ current have been characterized according to their pharmacology, voltage, and Ca2+ dependency and kinetic properties, namely transient outward (IA), delayed rectifying (IK) and inward rectifying (Kir) currents (12, 13, 14). IA is evoked at holding potentials more negative than –70 mV, is closed at potentials above –40 mV, and displays rapid inactivation. The IA current in rat somatotropes is kinetically similar to that seen in bovine somatotropes, rat lactotropes, and the GH3 somatotrope cell line (10, 15, 16) The IA channel antagonist 4-aminopirimidine (4-AP) stimulates GH secretion in ovine somatotropes, indicating that IA channels may be essential for maintaining the resting membrane potential and determining [Ca2+]i (13). IK is activated at around –20 mV from a holding potential of –40 mV and is slowly inactivating (13). Kir channels play an important role in maintaining resting membrane potential (14) but contribute only a small portion of total K+ currents in somatotropes (17).
The inhibitory effect of SRIF could be explained, at least partially, by the fact that SRIF increases K+ currents so that the frequency and duration of action potentials are reduced, which subsequently leads to the reduction in Ca2+ influx and GH release (4, 8, 12). In the present study, we tested the effect of SSTR subtype-specific agonists on voltage-gated K+ currents to clarify the subtype(s) of SSTRs involved in the increase in the K+ currents by SRIF in somatotropes.
Materials and Methods
Chemicals
Culture medium and fetal calf serum were purchased from Thermo Electron Corp. (Melbourne, Australia). Penicillin-streptomycin Fungizone antibiotic solution and trypsin were from Life Technologies, Inc. (Gaithersburg, MD). SSTR1–5-specific agonists were provided by Merck Research Laboratories. SRIF was purchased from Auspep (Melbourne, Australia), and tetrodotoxin, charybdotoxin (ChTX), and apamin were purchased from Alomone Laboratory (Jerusalem, Israel). Tetraethylammonium chloride (TEA), 4-AP, nifedipine, nystatin, dimethyl sulfoxide (DMSO), and all general salts for recording solutions were purchased from Sigma (St. Louis, MO).
Multiplex RT-PCR of SRIF receptor subtypes
PCR primer sequences for each of the rat SSTR subtypes were as follows: SSTR1, sense, 5'-CTA CTT TGC CGC CTG GTG CTC-3', and antisense, 5'-TGG CAA TGA TGA GCA CGT AAC-3' [GenBank accession no. (ACC) X62314]; SSTR2, sense, 5'-TTG ACG GTC ATG AGC ATC G-3', and antisense, 5'-ACA GAC ACG GAC GAG ACA TTG-3' (ACC M93273); SSTR3, sense, 5'-GGC CGC TGT TAC CTA TCC TTC-3', and antisense, 5'-GGC ACT CCT GAG AAC ACA ACC-3' (ACC X63574); SSTR4, sense, 5'-CGG AGA CGC TCA GAG AAG AAG-3', and antisense, 5'-TGG TCT TGG TGA AAG GGA CTC-3' (ACC M96544); SSTR5, sense, 5'-CAT GAG TGT TGA CCG CTA CC-3', and antisense, 5'-GGC ACA GCT ATT GGC ATA AG-3' (ACC L04535). The expected sizes of PCR products were 364 bp for SSTR1, 449 bp for SSTR2, 555 bp for SSTR3, 409 bp for SSTR4, and 508 bp for SSTR5. The SSTR subtype mRNA levels were obtained by RT-PCR, and all the procedures were taken from Park et al. (18). We have confirmed expression of SSTR1–5 in GH3 cells by RT-PCR. All PCRs were performed in a 25-μl volume containing 2 μl reverse transcription reaction, 2x Master Mix buffer (consisting of 3 mM MgCl2; 400 μM deoxyATP, deoxyGTP, deoxyCTP, deoxythymidine triphosphate; and 50 U/ml Taq DNA polymerase) (Promega Corp., Madison, WI). The thermal cycling profile was as follows: 95 C for 10 min, 24 (SSTR2, SSTR5) or 28 (SSTR1, SSTR3, SSTR4) cycles of 95 C for 30 sec, 60 C for 1 min, and 72 C for 1 min. The final extraction was at 72 C for 10 min. Reaction products were separated on a 1.4% gel and analyzed using the Quantity One gel program (Gel-DOC 2000; BioRad, Segrate, Italy).
Cell culture and preparation
The GH3 cell line (American Type Culture Collection, Manassas, VA), originally obtained from the rat pituitary, secretes prolactin and GH and secretion is regulated by GHRH and SRIF. The GH3 cells were grown as monolayers in 80-cm2 plastic culture flasks (Nunc Brand Product, Roskilde, Denmark) containing culture medium in a humidified atmosphere containing 5% CO2 at 37 C. The culture medium, which is composed of 44.5% DMEM, 44.5% Hams F-12, 10% fetal calf serum, and 1% penicillin streptomycin, was changed every 2 d. 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 d 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. Transmembrane currents were recorded using the patch-clamp technique in nystatin-perforated, whole-cell recording (WCR) configuration. Electrodes were pulled by a Sutter P-87 microelectrode puller (Sutter Equipment Co., Navato, CA) from borosilicate micropipettes with inner filament (Harvard Apparatus Ltd., Edenbridge, UK). After fire polishing, these electrodes had an initial input resistance of 3–5 M. All recordings were made using the Axopatch-1 C amplifier (Axon Instrument, Foster City, CA).
The bath solution contained 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4, adjusted with NaOH; osmolarity of 310 mOsm/liter). To exclude the occurrence of Ca2+ and Na+ currents, cobalt (Co2+, 3 mM), and tetrodotoxin (1 μM) were added to the bath solution on the day of experimentation.
The pipette solution contained 55 mM KCl, 75 mM K2SO4, 8 mM MgSO4, and 10 mM HEPES (pH 7.4 and osmolarity of 310 mOsm/liter), and the electrode was backfilled with this solution containing nystatin (300 μg/ml in 0.1% DMSO). This concentration of DMSO, applied to the cell studied in the WCR configuration, had no effect on membrane conductance.
Cell culture dishes were fixed on the stage of an inverted microscope (Olympus, New Hyde Park, NY). After obtaining a high-resistance seal using a micromanipulator (Narishige, Tokyo, Japan), the pipette potential was held at –70 mV and voltage pulses (10 mV, 200-msec 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 forming a seal. Whole-cell capacitance and series resistance (using only cells with <35 M) were compensated (>80%) before experimentation, and leak current was routinely subtracted using the Clampex 8.0 program (Axon Instruments). The change in series resistance over the course of each experiment was also monitored, and recordings with significant change (more than 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.
Each SSTR-specific agonist, SRIF, and K+ channel blockers (ChTX, apamin, 4-AP, TEA) were applied by a gravity pressure perfusion system at a rate of approximately 1 ml/min. Control recordings were started at least 10 min after patching the cell. 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 K+ currents in our recording system. All experiments were performed at room temperature (20–22 C).
Varying the concentration of SSTR 1–5
The specific agonists for SSTR1–5 used in this study were: L-797,591 (SSTR1), L-779,976 (SSTR2), L-796,778 (SSTR3), L-803,087 (SSTR4), and L-817,818 (SSTR5). To find the maximal response concentration, a range of doses were tested. For SSTR1 and SSTR5, 10–9, 10–8, and 10–7 M were tested, and for SSTR2 and SSTR4, 10–10, 10–9, 10–8, and 10–7 M were tested. For SSTR3, only 10–8 and 10–7 M were tested. The final concentration of each agonist that we used as maximal was 10–8 M and submaximal as 10–9 M. The final concentration of SRIF was 200 nM.
Data analysis
The pCLAMP 8.0 software (Axon Instruments) was used to record and analyze the data. Effect of SSTR agonists was tested using 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 mean ± SEM. The example traces displayed were chosen as representative of at least six recordings under the same experimental conditions unless otherwise indicated in the text.
Results
Multiplex RT-PCR for pituitary SSTR subtypes
We confirmed expression of SSTR1–5 in GH3 cells by multiplex RT-PCR (Fig. 1). The multiplex RT-PCR assay in this study amplified SSTR2 and SSTR5 or SSTR1, SSTR3, and SSTR4 transcripts from GH3 cell culture in a single tube, resulting in PCR products of the expected sizes (Fig. 1, A and B).
Characterization of the voltage-gated K+ current in GH3 cells
In the presence of blockers of Ca2+ and Na+ channels as detailed in Materials and Methods, voltage-gated K+ currents were recorded using nystatin-perforated WCR. To test for the presence of Ca2+-activated K+ currents (KCa), ChTX, a blocker of large conductance KCa channels, and apamin, a specific blocker of small conductance KCa channels, were applied to cells. Local application of ChTX (1 μM) or apamin (1 μM) did not modify the recorded K+ currents (results not shown). Addition of SRIF in the presence of ChTX and apamin increased voltage-gated K+ current amplitude significantly (results not shown) to the same level as SRIF alone, thus excluding the involvement of KCa currents in our experiments. K+ currents were evoked by voltage steps from a holding potential of either –70 mV (total currents) or –40 mV (IK currents only) to potentials of –50 to +60 mV, in increments of 10 mV over 200 msec (Fig. 2). The GH3 cells possessed both IA and IK currents. Only the IK current was recorded during depolarizations from a holding potential of –40 mV (Fig. 2Ab), whereas the IA (Fig. 2Ac) was revealed when this IK current was subtracted from total currents recorded with a holding potential of –70 mV (Fig. 2Aa). The current-voltage relationship for total K+ currents (IA and IK, Fig. 2Aa), the IK current (Fig. 2Ab), and the IA current (Fig. 2Ac) are shown in Fig. 2B. It is obvious that the IA current is a small component of total K+ currents in GH3 cells, compared with a large component attributable to the IK current.
In addition to different voltage-dependent characters, IA and IK channels have different sensitivities to some K+ channel antagonists. The IA channel is highly sensitive to 4-AP, whereas the IK channel can be blocked by TEA (12). As shown in Fig. 3, total K+ current was recorded from cells at a holding potential of –70 mV (Fig. 3Aa), whereas the transient component was absent in the presence of 4-AP (3 mM) (Fig. 3Ab). This result indicates an inhibition of predominantly the IA current. The current that persisted in the presence of 4-AP was predominantly the IK current, which was completely blocked by TEA (10 mM) (Fig. 3Ad). The current-voltage relationships of total peak current (IA and IK, Fig. 3Aa) and IK currents (Fig. 3Ab) are shown in Fig. 3B and are similar to the data obtained with different holding potentials in Fig. 2. The difference between these two traces (Fig. 3Ac) reveals the total 4-AP-sensitive IA current and is similar to the IA current revealed by the difference in currents obtained at holding potentials of –70 vs. –40 mV (Fig. 2Ac).
Time response of SRIF effects
To investigate the time course of the K+ current response to SRIF, total voltage-gated K+ currents were recorded with single steps from a holding potential of –70 to +60 mV every minute. The SRIF-induced increase in K+ currents occurred 2–3 min after application and reached a maximum after 10–12 min and recovered completely 10–15 min after removal of SRIF (Fig. 4).
SRIF activates the voltage-gated K+ current
Application of SRIF (200 nM) significantly increased the total voltage-gated K+ currents evoked by depolarizing steps from a holding potential of –70 mV (Fig. 5). SRIF application increased both IA and IK currents without preference because both the peak (IA and IK) and sustained (mostly IK) components of the response were enhanced equivalently (in Fig. 5, compare A1 and A2). In addition, current-voltage relationships of K+ currents before and after application of SRIF indicate that there is no kinetic change with the application of SRIF, although the amplitude of total currents at many depolarizing steps is increased (Fig. 5C). The effect was reversible, with total recovery of peak current observed 10 min after the removal of SRIF (Fig. 5A3). Mean (±SEM) values of current measured at a step to +60 mV in eight cells are shown in Fig. 5B and increase in the K+ currents by SRIF was statistically significant (P < 0.01). Both IA and IK currents were increased by SRIF (Fig. 6, A and B). The mean (±SEM) increase in IA and IK current amplitude at +60 mV by SRIF measured from four cells was statistically significant (Fig. 6C; P < 0.05).
Effect of different doses of SSTR agonists on K+ current
The dose-dependent increase in K+ current by SSTR agonists was carried out from a holding potential of –70 mV. Mean amplitude of the K+ current at +60 mV for each agonist is shown in Fig. 7. SSTR1 and SSTR5 agonists had no effect at 10–9 M, whereas 10–8 and 10–7 M significantly increased [P < 0.05 (SSTR1), P < 0.01 (SSTR5)] K+ current amplitude (Fig. 7, A and E). SSTR2 and SSTR4 agonists at 10–10 M did not affect K+ current amplitude, but exposure to 10–9 and 10–8 M increased it significantly (P < 0.01) over control, whereas 10–7 M of these agonists had no further effect. The SSTR3 agonist, at 10–8 or 10–7 M, did not affect K+ current amplitude, compared with control (Fig. 7C). It is therefore clear that 10–8 M is the maximal effective concentration for each agonist, apart from the SSTR3 agonist, whereas10–9 M activates the voltage-gated K+ currents to submaximal levels.
Effect of SSTR subtype-selective agonists on K+ current
The maximal concentration of each SSTR subtype agonist (10–8 M) was applied, with a subsequent application of SRIF and the effects on total K+ current from eight cells is shown in Fig. 8. SSTR1 and SSTR5 agonists increased K+ current significantly (P < 0.01), and addition of SRIF in the presence of these agonists further increased the K+ current significantly (Fig. 8, A and E). SSTR2 and SSTR4 agonists increased K+ current significantly (P < 0.01), but further addition of SRIF in the presence of these agonists had no additional effect on K+ current (Fig. 8, B and D). The SSTR3 agonist itself had no effect on the K+ current, but SRIF application in the presence of SSTR3 agonist increased the K+ current significantly (P < 0.01, Fig. 8C).
Because the SSTR1 agonist, L-797,591, can activate SSTR4 receptors, albeit at 100 times lower affinity than for SSTR1, and the SSTR5 agonist, L-817,818, showed 8 times higher affinity for SSTR5, compared with SSTR1, and 130 times higher than SSTR2 (5), the partial increase in the K+ current by L-797,591 and L-817,818 may reflect a cross-reaction of other SSTRs subtypes rather than on SSTR1 and SSTR5 specifically. Submaximal concentration, 10–9 M, of agonists was therefore tested followed by exposure to SRIF. SSTR2 and SSTR4 agonists at 10–9 M increased the K+ current significantly (P < 0.01), and addition of SRIF further increased the K+ current significantly (P < 0.05 and P < 0.01, Fig. 9, B and C). Although SSTR1 and SSTR5 agonists 10–9 M had no effect on the K+ current, addition of SRIF in the presence of these agonists increased the K+ current fully (P < 0.01, Fig. 9, A and D).
Discussion
Five subtypes of SSTRs have been cloned and identified from various species including human, rat, mouse, porcine, and bovine (19, 20). Binding of SRIF or SRIF ligands to SSTRs induces G protein activation and signaling through various pathways, resulting in activation of K+ channels and consequent suppression of voltage-gated Ca2+ influx (21, 22).
In this present study, we examined which subtype or subtypes of SSTR are involved in the modification of the voltage-gated K+ current by SRIF in GH3 cells using SSTR subtype agonists. Activation of SSTR3 has no effect on K+ current, ruling out its involvement in the SRIF-induced responses. Full activation of SSTR2 or SSTR4 increased the amplitude of the K+ current to the same level as that of SRIF alone. In contrast, SSTR1 and SSTR5 agonists produce only a partial increase in the K+ current. This partial activation of K+ current by the maximal concentration of SSTR1 and SSTR5 agonists may not actually be due to the activation of SSTR1 and -5 because these agonists have a moderate affinity for either SSTR2 or SSTR4 as well (5). A submaximal dose of agonists for SSTR1 and SSTR5 induced no effect on the K+ current, whereas the same dose of agonists for SSTR2 and SSTR4 evoked significant increase in the same K+ current. It is therefore possible that SSTR1 and SSTR5 may not be involved in the SRIF-induced increase in voltage-gated K+ currents due to the potential nonspecific effects of SSTR1 and -5 agonists on SSTR2 and -4. Whereas we cannot be completely certain of this conclusion from our current experiments, we can be certain that stimulation of either SSTR2 or -4 can fully activate K+ currents with no further increase by SRIF.
One possible explanation for this is that activation of either SSTR2 or SSTR4 alone is capable of fully activating the signal transduction system, which is responsible for the increase in voltage-gated K+ currents by SRIF. Thus, as long as one receptor type, either SSTR2 or SSTR4, is fully activated, maximal SRIF-induced increment of the K+ current can be achieved. Another possibility is that SSTR2 and SSTR4 undergo heterodimerization in GH3 cells. Recent studies have shown that SSTRs exhibit SRIF-induced homo- and heterodimerization, which changes their binding and functional properties (23, 24, 25). For example, heterodimerization of SSTR2 and SSTR3 in human embryonic kidney cells resulted in high-affinity binding to SRIF and SSTR2 agonist (L-779,976) but not to the SSTR3 agonist (L-797,778) (26). SSTR2 is most abundantly expressed in neural and neuroendocrine tissues (27), especially in the rat (28). In the rat pituitary, the rank of order of expression of SSTRs is SSTR2 > SSTR1 = SSTR3 > SSTR5 > SSTR4 (27, 29, 30). Although SSTR4 is expressed at high levels in the habenula and heart of rat (30, 31, 32), it is the least expressed SSTR in the pituitary of that species. Thus, activation of either SSTR2 or SSTR4 may induce dimerization, and this dimer then activates the voltage-gated K+ channels to increase K+ currents. The role of SSTR heterodimerization, if true, will have a major impact on future drug design (33).
Based on kinetics, voltage dependency, and pharmacological sensitivities, four major types of voltage-gated K+ currents identified in mammalian pituitary cells are Kir, IA, IK, and M-type K+ currents (10, 12, 13, 21). In different species there may be different proportions of each type of voltage-gated K+ channel. For example, IA makes up a large proportion of the total K+ current in rat pituitary cells but accounts for only a small component of the total K+ current in sheep somatotropes (12, 34). From our experiments, the majority of voltage-gated K+ currents were composed of IK currents, with a small proportion being IA current. We have demonstrated these two types of voltage-gated K+ currents based on their voltage-dependent kinetics and pharmacological characteristics.
Previous work on GH3 cells indicated low levels of two types of Ca2+-activated K+ currents (35). In the present study, Ca2+-activated K+ currents in GH3 cells seem to be absent under the recording conditions used in the presence of the Ca2+ channel blocker, Co2+ (36). The K+ current response to SRIF reported here does not involve Ca2+-activated K+ currents.
The results from our study demonstrate that voltage-gated K+ currents in GH3 cells are significantly and reversibly increased by application of SRIF. This result is in agreement with several reports that SRIF increases K+ current in primary cultured rat and ovine somatotropes (12, 13, 34). The functional consequence of this increase in K+ current would be to hyperpolarize the plasma membrane (10, 13, 37). This would reduce the probability of opening of voltage-gated Ca2+ channels and hence reduce the action potential duration and hence reduce [Ca2+]i and GH secretion.
In conclusion, using selective SSTR2 and -4 agonists, we have demonstrated that SSTR2 and SSTR4 are involved in the SRIF-induced increase in voltage-gated K+ currents in GH3 cells. This may be possible if stimulation of either SSTR2 or SSTR4 alone can fully activate the signal transduction system(s) responsible for the increase in K+ currents by SRIF. Alternatively, SSTR2 and SSTR4 may undergo heterodimerization; however, both these possibilities require further investigation. Activation of the voltage-gated K+ current by SRIF is likely to decrease the level of [Ca2+]i and GH secretion as a result of membrane hyperpolarization and a decrease in action potential frequency and/or duration.
Acknowledgments
We thank Ms. M. Hernandez and D. Feng for their help on cell preparation, Ms. D. Arnold for manuscript editing, and Ms. S. Panckridge for graphical preparation.
Footnotes
This work was supported by Australian National Health and Medical Research Council.
Abbreviations: 4-AP, 4-Aminopirimidine; [Ca2+]i, intracellular-free Ca2+; ChTX, charybdotoxin; DMSO, dimethyl sulfoxide; IA, transient outward K+ current; IK, delayed rectifying K+ current; KCa, Ca2+-activated K+ current; Kir, inward rectifying current; SRIF, somatostatin; SSTR, SRIF receptor; TEA, tetraethylammonium chloride; WCR, whole-cell recording.
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