Endocrinology Vol. 143, No. 7 2659-2663
Copyright © 2002 by The Endocrine Society
The Effect of Two-Day Treatment of Primary Cultured Ovine Somatotropes with GHRP-2 on Membrane Voltage-Gated K+ Currents
Chen Chen
Department of Endocrine Cell Biology, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: Chen Chen, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: . chen.chen{at}med.monash.edu.au
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Abstract
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Long-term in vivo treatment with synthetic GH-releasing peptides (GHRPs) enhances the release of GH induced by endogenous GHRH. The mechanism for such an enhancement on GH release is unknown. In this experiment, somatotropes were obtained from ovine pituitaries by enzyme dissociation and enriched by density centrifugation. Membrane voltage and currents were recorded with whole-cell patch-clamp configuration. After 48-h treatment with GHRP-2 (10-8 M), the percentage of cells with spontaneous action potential was increased (51 vs. 27%) without change of resting potential. This GHRP-2 treatment also increased the amplitude of voltage-gated K+ currents (predominantly transient A-type-like current but also delayed rectifier or K-type-like current) without modification of biophysical kinetics. Down-regulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate at the time of adding GHRP-2 blocked the increase in K+ currents. Inclusion of calphostin C (PKC inhibitor) but not H89 (protein kinase A inhibitor) significantly reduced the increase in K+ currents by GHRP-2. Inclusion of actinomycin D (transcription inhibitor) or cycloheximide (protein synthesis inhibitor) abolished the increase in K+ currents. These data indicate that 48-h GHRP-2 treatment increases the density of K+ channels via PKC and channel protein synthesis. Such a modification on K+ channels by GHRP-2 may be partially responsible for the change of somatotrope electrophysiological properties and sensitivity to GHRH stimulation.
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Introduction
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GHRH IS RELEASED FROM nerve terminals in the median eminence and stimulates GH secretion from somatotropes (1). Synthetic GH-releasing peptides (GHRPs) have also been shown to elevate GH levels in vivo and in vitro (2). The chemical structure of GHRPs and nonpeptidergic GH-secretagogues (such as MK-677, which mimics the three-dimension structure of GHRP-6) provides a series of useful and economical drug candidates to elevate GH levels. Although GHRP has been reported to specifically stimulate GH release in a number of species of animals (3, 4), available data also indicate a synergistic effect of GHRPs with endogenous GHRH in vivo (5). In terms of stimulating GH secretion in vitro, a recently synthesized GHRP (GHRP-2) with a structure ofD-Ala-D-ßNal-Ala-Trp-D-Phe-Lys-NH2, has been demonstrated to stimulate GH secretion in a much more potent manner than earlier versions of GHRP in humans and animals (6, 7). Treatment with GH secretagogues increases pulsatile GH secretion in vivo and enhances the sensitivity of somatotropes to GHRH (8), which indicates a possible priming effect of GHRP on somatotropes. The mechanism by which GHRP exerts these effects on the pituitary gland is not clear, although pituitary-specific transcription factor expression in rat somatotropes is increased by GHRP-6 (9).
Somatotropes are excitable cells and exhibit spontaneous action potential (10). Treatment of somatotropes with GHRH depolarizes cell membrane potential and stimulates Ca2+ influx via transmembrane Ca2+ channels leading to an increases in the level of intracellular free Ca2+ ([Ca2+]i) (11). Modification of cell electrophysiological properties in somatotropes will lead to a change in sensitivity to GHRH. The K+ conductance is the predominant membrane ion conductance at resting membrane potential, which regulates cell excitability. It is therefore necessary to study the effect of GHRP on transmembrane K+ current in somatotropes in vitro.
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Materials and Methods
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Cell culture and GHRP-2 treatment
Sheep pituitaries were obtained from a local abattoir and then subjected to collagenase/pancreatin treatment to liberate cells as described previously (12). More than 3 x 107 cells were usually obtained from one pituitary gland, with greater than 90% viability (trypan blue exclusion test). The cell suspension (3–5 ml) was then placed, under sterile conditions, above a layered column of the Percoll Fractions of increasing density and centrifuged as described previously (12). Fractions (1 and 2), which contained up to 85% of somatotropes, were plated onto 35-mm Petri dishes to grow as monolayer and used in these experiments. In each case, penicillin/streptomycin was used in the culture medium for the first 24 h. The culture medium was then replenished every 2 d using DMEM supplemented with 10% sheep serum, 2% fetal calf serum, and 1% (vol/vol) L-glutamine (200 mM).
After 3–5 d in vitro in a humidified incubator (37 C, air 95%, CO2 5%), 10 µl GHRP-2 stock solution or vehicle were added to each treatment or control dish every 12 h for a total of 48 h before electrophysiology recording. The final concentration of GHRP-2 in the 2-ml culture medium was 10-8M.
Electrophysiological recording
On the day of recording, culture medium was replaced by patch-clamp bath solution 30 min before recording. Transmembrane voltage and currents were recorded using the gigaseal patch-clamp technique in conventional whole-cell recording configuration (13). Electrodes were pulled by a Sutter P-87 microelectrode puller from borosilicate micropipettes with inner filament and had an initial input resistance of 5–8 M
after fire polishing. Recordings were made on the stage of an inverted microscope (Olympus Corp., Shibuya-ku, Tokyo, Japan). When transmembrane voltage was recorded, the standard bath solution was composed of the following (in millimoles): 140 NaCl, 5 KCl, 2.5 CaCl2, 0.5 MgCl2, 10 glucose, and 10 HEPES at pH 7.4 and osmolarity of 310 mOsm. When the voltage-gated K+ current was isolated, the bath solution was composed of the following (millimoles): NaCl (140), CaCl2 (0.5), CdCl2 (1), KCl (5), MgCl2 (0.5), HEPES (10), glucose (10), and 1 µM tetrodotoxin at pH 7.4 and osmolarity of 310 mOsm. The electrode solution was composed of the following (millimoles): KCl (140), EGTA (10), MgCl2 (1), HEPES (10), and glucose (10). An ATP regenerative system (2 mM ATP, 5 mM Na2-phosphocreatine, and 20 U/ml creatine phosphokinase) was added into the electrode solution just before recording, and the electrode solution was then adjusted to pH 7.4 and osmolarity of 300 mOsm.
Chemicals
Culture media were obtained from Cytosystems (Castle Hill, Australia); sera, L-glutamine, and pancreatin were from Life Technologies, Inc. (Gaithersburg, MD); collagenase was obtained from Worthington Biochemical Corp. (Freehold, NJ); and DNase and all salts for experimental solutions were purchased from Sigma (St. Louis, MO).
Data analysis
See Fig. 2
, showing K+ current traces that are representative examples from a group of experiments. To exclude the influence of cell size, the K+ current value for statistic analysis was calculated as current (picoAbsorbance) recorded on one unit membrane capacitance (picofarad). The group of data is presented as the mean (± SEM) of at least six separate experiments with the same treatment protocol. All experiments conformed to the National Health and Medical Research Council (Australia) ethics code of practice. Effects of treatments were considered significant at P < 0.05 level using the ANOVA test followed by the Dunnett post hoc test.
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Figure 2. Effects of 2-d treatment with GHRP-2 on the voltage-gated K+ currents in ovine somatotropes. A, Voltage-gated K+ currents were evoked by depolarizing pulses every 2 sec with a 10-mV interval from a holding potential of -80 mV up to +60 mV as indicated at the bottom of the panel. Top traces show the K+ currents recorded from cell cultures treated with vehicle representing the K+ currents under control conditions. Middle traces show the K+ currents recorded from cell cultures treated with 10 nM GHRP-2 representing the K+ currents in GHRP-2-treated condition. GHRP-2 treatment increased amplitude of the K+ currents and the proportion of the transient K+ current among total currents. B, Current-voltage relationships of recorded K+ currents from control and GHRP-2-treated groups of somatotropes (n = 12 for each group). To reflect the proportional change of the transient component in total currents, current values were measured at 20-msec (transient) and 200-msec (mixed transient and delayed rectifying) time points after start of the depolarizing pulses. The current value was then divided by membrane capacitance, which is proportional to the total membrane area. This calculated value was appropriate to serve as an index for the density of the voltage-gated K+ channels on the membrane of somatotropes.
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Results
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The effect of GHRP-2 on membrane electrophysiology properties
Somatotropes like other pituitary endocrine cells are excitable cells with occasional spontaneous action potential (14, 15). Under current-clamp condition with the standard bath solution of 140 mM of Na+ and 2.5 mM of Ca2+ and pipette solution of 140 mM K+, membrane resting potential (RP) and spontaneous action potential (SAP) was recorded when pipette current was held to 0 for 5–10 min. In four replicate cultures, treatment of cells with GHRP-2 (10-8 M) for 48 h slightly hyperpolarized cell membrane RP but increased the percentage of cells with SAP (Fig. 1).
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Figure 1. Effect of 2-d GHRP-2 treatment on RP and SAP in ovine somatotropes. A, Percentage of cells with SAP was increased from 27% to 51% by 2-d treatment of cells with 10 nM GHRP-2 in culture medium. B, Resting membrane potential was marginally more negative in somatotropes treated with GHRP-2 (10 nM) for 2 d than that in control cultures. The difference is, however, no statistically significant.
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The effect of GHRP-2 on membrane voltage-gated K+ currents
To isolate the voltage-gated K+ currents, cells were perfused with low Ca2+ bath solution in the presence of 1 mM Cd2+ and 1 µM tetrodotoxin, outward K+ currents were triggered by depolarizing voltage steps from a holding potential of -80 mV. Current amplitude was measured in the test voltage range from -50 mV to +60 mV, with an increment of 10 mV between steps (Fig. 2A
). As we showed previously, there were two major types [transient A-type-like current (IA) and delayed rectifier or K-type-like current (IK)] of voltage-gated K+ currents in ovine somatotropes, with IK being the dominant type (16). Treatment of cells with GHRP-2 (10-8 M) for 48 h increased amplitude of K+ currents, which was more pronounced for the transient IA K+ current (Fig. 2A). On the basis of the kinetics of voltage-gated K+ currents investigated previously in this laboratory (16), the amplitude of currents measured at a time point of 20 msec after start of depolarizing pulses reflected a mixture of both IA and IK, whereas the current measured at the end of 200 msec depolarizing pulses reflect only the IK component. The GHRP-induced increase in the K+ currents measured at 20 msec was more significant than that measured at 200 msec after the depolarizing pulse (Fig. 2B). From a group of 12 cells, it was clear that the increase in the K+ currents measured at 20 msec was statistically more significant than that measured at 200 msec point during a depolarizing pulse from the holding potential to +60 mV (Fig. 3), which suggested that the increase in IA was more pronounced than the increase in IK after the GHRP-2 treatment.
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Figure 3. Effects of GHRP-2 treatment on IA and IK K+ currents in ovine somatotropes. The value of K+ currents was taken when membrane potential was depolarized from a holding potential of -80 mV to +60 mV. In the GHRP-2-treated group (n = 12), the K+ currents measured at both 20- and 200-msec time points during depolarizing pulses were significantly increased with more pronounced enhancement at 20 msec than that at 200-msec time point. *, P < 0.05; **, P < 0.01.
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Role of protein kinases A and C
As demonstrated previously, GHRP-2 stimulated protein kinase C (PKC) translocation from cytosol to membrane (17) and increased intracellular cAMP levels and protein kinase A (PKA) activity (18) in ovine somatotropes. The involvement of cAMP/PKA or PKC was investigated in this experiment. H89 (0.5 µM), a specific PKA inhibitor, did not modify voltage-gated K+ currents by itself and did not inhibit the increase in the K+ currents by GHRP-2 treatment when added to the culture medium at the same time with GHRP-2 (Fig. 4A). Phorbol 12-myristate 13-acetate (PMA) was used to down-regulate the PKC system in the cells by treating cell cultures 12 h before addition of GHRP-2. PMA (2 µM) down-regulation of the PKC did not significantly regulate the level of voltage-gated K+ currents but significantly reduced the increase in the K+ currents by GHRP-2 treatment (P < 0.01, compared with the group treated with GHRP-2 alone, Fig. 4B). In addition to PMA down-regulation, a specific PKC inhibitor, calphostin-C (100 nM), also reduced the K+ current enhancement by GHRP-2 treatment, although no change of basal K+ current was observed by calphostin C treatment alone (P < 0.05, Fig. 4C). These data indicate the requirement of the PKC system in the cells for the K+ current increase by 48-h GHRP-2 treatment.
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Figure 4. Involvement of PKA and PKC in the effect of GHRP-2 treatment on the K+ currents. A, K+ currents were evoked by depolarizing pulses from a holding potential of -80 mV to +60 mV and measured at 20- and 200-msec time points of the depolarizing pulses. Data shown in this figure represent mean (±SEM) of the measured K+ currents (n = 10). Addition of H89 (0.5 µM, PKA inhibitor) did not change the amplitude of the K+ currents and did not alter the increase in the K+ currents by GHRP-2 treatment. There was no difference between groups treated with GHRP-2 alone and GHRP-2 + H89. *, P < 0.05; **, P < 0.01, compared with control and groups treated with H89 alone. B, K+ currents were evoked by depolarizing pulses from a holding potential of -80 mV to +60 mV and measured at 20- and 200-msec time points of the depolarizing pulses. Data shown in this figure represent mean (±SEM) of the measured K+ currents (n = 10). Addition of PMA (2 µM, PKC down-regulation) did not change the amplitude of the K+ currents but totally blocked the increase in the K+ currents by GHRP-2 treatment. There was a significant difference between groups treated with GHRP-2 alone and GHRP-2 + PMA. *, P < 0.05; **, P < 0.01, compared with control groups and groups treated with PMA alone and PMA + GHRP-2. C, K+ currents were evoked by depolarizing pulses from a holding potential of -80 mV to +60 mV and measured at 20- and 200-msec time points of the depolarizing pulses. Data shown in this figure represent mean (±SEM) of the measured K+ currents (n = 10). Addition of calphostin C (100 nM, PKC inhibitor) did not change the amplitude of the K+ currents but totally blocked the increase in the K+ currents by GHRP-2. There was a significant difference between groups treated with GHRP-2 alone and GHRP-2 + calphostin C. *, P < 0.05; **, P < 0.01, compared with control groups and groups treated with calphostin C alone and calphostin C + GHRP-2.
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Role of RNA transcription and protein synthesis in the K+ current response to GHRP-2
Because there was no significant alteration of the K+ current kinetics after GHRP-2 treatment, it was assumed that K+ channel numbers were increased by this long-term GHRP-2 treatment. Actinomycin D, an inhibitor for mRNA transcription, was used in culture medium at the same time that GHRP-2 was added. Actinomycin D (4 µM) totally blocked the increase in the K+ current by GHRP-2 (Fig. 5A). Cycloheximide, an inhibitor of protein synthesis, was also tested in cell cultures in the same time period of GHRP-2 treatment. Cycloheximide (50 µM) significantly reduced the recorded K+ currents and also completely abolished the increase in K+ current by 48-h GHRP-2 treatment (Fig. 5B).
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Figure 5. Effect of protein synthesis inhibitors on the GHRP-2-induced increase in the K+ currents. A, K+ currents were evoked by depolarizing pulses from a holding potential of -80 mV to +60 mV and measured at 20- and 200-msec time points of the depolarizing pulses. Data shown in this figure represent mean (±SEM) of the measured K+ currents (n = 8). Addition of actinomycin D (4 µM, transcription inhibitor) did not significantly change the amplitude of the K+ currents but totally blocked the increase in the K+ currents by GHRP-2 treatment. There was a significant difference between groups treated with GHRP-2 alone and GHRP-2 + actinomycin D. **, P < 0.01, compared with control, actinomycin C alone, and actinomycin D + GHRP-2-treated groups. B, K+ currents were evoked by depolarizing pulses from a holding potential of -80 mV to +60 mV and measured at 20- and 200-msec time points of the depolarizing pulses. Data shown in this figure represent mean (±SEM) of the measured K+ currents (n = 8). Addition of cycloheximide (50 µM, protein synthesis inhibitor) significantly reduced the amplitude of the K+ currents and totally blocked the increase in the K+ currents by GHRP-2 treatment. *, P < 0.05; **, P < 0.01, compared with control group.
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Discussion
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This experiment provides convincing evidence that long-term (48 h) treatment in vitro with GHRP-2 increased excitability of ovine somatotropes and the levels of voltage-gated K+ currents. Such an increase in the voltage-gated K+ currents requires a normal function of the PKC system and is achieved through the increase in the K+ channel protein synthesis.
Somatotropes, like most other pituitary endocrine cells, are excitable cells with irregular spontaneous action potential (12). Cell excitability depends on the properties of transmembrane ion channels and the level of resting membrane potential. The resting membrane potential is built mainly on a selective permeability to K+ ion. If the membrane permeability to K+ ion increases, the resting membrane potential is hyperpolarized. The depolarizing phase of an action potential is composed mainly of the voltage-gated inward Ca2+ and Na+ currents (14). As demonstrated previously, a resting membrane potential between -40 and -60 mV is the voltage range with maximal variety of the availability of the voltage-gated Ca2+ and Na+ channels because of their voltage-dependent inactivation properties (14, 19). A hyperpolarization in the range of -40 and -60 mV increases the availability of voltage-gated Ca2+ and Na+ channels, leading to an increase in the Ca2+ and Na+ inward currents and the cell excitability (14, 19). Such a slight hyperpolarization may enhance the GHRH-induced membrane depolarization and increase in action potential frequency in ovine somatotropes (11) through the increase in Ca2+ and Na+ channel availability. This enhancement of GHRH stimulation may also express an increase in GHRH-stimulated GH secretion. Through the same mechanism, the slight hyperpolarization observed in GHRP-2-treated cells may also contribute to the increase in the percentage of cells with spontaneous action potential.
The other factor, which may contribute to the action potential frequency, is the voltage-gated K+ currents. Ovine somatotropes have mainly two types of voltage-gated K+ currents, IA and IK. Both currents have been demonstrated to be involved in membrane electrical activity of somatotropes (20). IA was thought to be partially responsible for maintaining the resting potential levels and to participate the repolarizing process of action potential (12). IK current can be activated by a group of continuously firing action potential, which may help the cell for subsequent action potential and also prevent overloading of free Ca2+ in the cell (21). Modification of these two voltage-gated K+ currents also can contribute to the alternation of cell excitability. An increase in action potential is directly linked to the levels of [Ca2+]i because every single action potential is enough to trigger a detectable increase in [Ca2+]i in single pituitary endocrine cell (22). The increase in action potential frequency will therefore increase the oscillation of [Ca2+]i and probably raise the basal level of GH secretion. In this experiment, both IA and IK currents were increased by GHRP-2 treatment, the action on IA current being the predominant one.
Signaling systems employed by GHRP-2 have been investigated previously. In terms of inducing GH secretion from ovine somatotropes, both the cAMP-PKA pathway and PKC pathway are involved in the response (4). Both PKA and PKC systems are activated by challenging the cells with GHRP-2 in vitro (17, 18). We therefore investigated whether these signaling pathways were involved in the increase in voltage-gated K+ currents in this experiment. PKC down-regulation at the same time of GHRP-2 treatment prevented the increase in the K+ currents. PKC specific inhibitor, calphostin C, also significantly diminished the increase in the K+ currents by GHRP-2 treatment. The specific inhibitor for PKA, H89, however, did not affect the basal and GHRP-2 induced increase in the K+ currents. As indicated previously, the major signaling systems responsible for the stimulation of GH secretion by GHRP-2 in ovine somatotropes was the cAMP-PKA system (4, 17, 18). The PKC has been demonstrated in this experiment as the major signaling molecule involved in the increase in K+ currents by 48-h treatment of cells with GHRP-2. It is therefore possible that the cAMP-PKA signaling system in ovine somatotropes is involved mainly in the short-term effect of GHRP-2 to stimulate GH secretion. The activation of PKC by GHRP-2 in ovine somatotropes may play an important role in the long-term effect of GHRP-2 to regulate membrane voltage-gated K+ channels and may also be responsible for the alteration of excitability of the somatotropes by GHRP-2 treatment.
The modification of K+ currents obtained in this experiment is reflected mainly by the increase in the amplitude of the currents. Activation kinetics of IA and IK were not significantly changed by GHRP-2 treatment, which therefore indicates a possible increase in K+ channel numbers or density on the cell membrane. We have used two different types of drugs to block the protein synthesis [cycloheximide (23)] or mRNA transcription [actinomycin D (24)]. Both blockers, especially the cycloheximide, reduced the K+ current density. In addition, the GHRP-2-induced increase in K+ currents was abolished by the treatment of cells with either cycloheximide or actinomycin D. The effect of cycloheximide is more significant than that of actinomycin D, which may reflect a highly efficient blockade of protein synthesis by this drug.
In summary, we conclude that the treatment of cells with GHRP-2 for 48 h increases the excitability of somatotropes and the voltage-gated K+ currents on the membrane. The increase in the K+ currents is achieved through an increase in the synthesis of K+ channels. In addition, normal function of PKC system is required for the increase in voltage-gated K+ currents by GHRP-2.
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Acknowledgments
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I would like to thank K. Loneragan and M. Hernandez for technical assistance, and S. Panckridge for preparing graphics. I also thank Prof. C. Y. Bowers at Tulane Medical Center (New Orleans, LA) for supplying synthetic GHRP-2 used in this experiment.
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Footnotes
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This work was supported by the Australian National Health and Medical Research Council.
Abbreviations: [Ca2+]i, Intracellular free Ca2+; GHRP, GH-releasing peptide; IA, transient A-type-like current; IK, delayed rectifier or K-type-like current; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RP, resting potential; SAP, spontaneous action potential.
Received December 10, 2001.
Accepted for publication March 26, 2002.
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Abstract
Introduction
