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Involvement of Tetrodotoxin-Resistant Na⁺ Current and Protein Kinase C in the Action of Growth Hormone (GH)-Releasing Hormone on Primary Cultured Somatotropes from GH-Green Fluorescent Protein Transgenic Mice

Prince Henry’s Institute of Medical Research (S.-K.Y., K.W., C.C.), Melbourne 3168, Victoria, Australia; and Department of Physiology (S.-K.Y., H.P.), Monash University, Melbourne 3004, Victoria, Australia

Address all correspondence and requests for reprints to: Chen Chen, M.D., Ph.D., School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. E-mail: chen.chen{at}uq.edu.au.

	Abstract

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GHRH depolarizes the membrane of somatotropes, leading to an increase in intracellular free Ca²⁺ concentration and GH secretion. Na⁺ channels mediate the rapid depolarization during the initial phase of the action potential, and this regulates Ca²⁺ influx and GH secretion. GHRH increases a tetrodotoxin-sensitive somatotrope Na⁺ current that is mediated by cAMP. TTX-resistant (TTX-R) Na⁺ channels are abundant in sensory neurons and cardiac myocytes, but their occurrence and/or function in somatotropes has not been investigated. Here we demonstrate expression of TTX-R Na⁺ channels and a TTX-R Na⁺ current, using patch-clamp method, in green fluorescent protein-GH transgenic mouse somatotropes. GHRH (100nM) increased the TTX-R Na⁺ current in a reversible manner. The GHRH-induced increase in TTX-R Na⁺ current was not affected by the cAMP antagonist Rp-cAMP or protein kinase A inhibitors KT5720 or H89. The TTX-R current was increased by 8-bromoadenosine-cAMP (cAMP analog), forskolin (adenylyl-cyclase activator), and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), but the additional, GHRH-induced increase in TTX-R Na⁺ currents was not affected. U-73122 (phospholipase C inhibitor) and protein kinase C (PKC) inhibitors, Gö-6983 and chelerythrine, blocked the effect of GHRH. PKC activators, phorbol dibutyrate and phorbol myristate acetate, increased the TTX-R Na⁺ current, but GHRH had no further effect on the current. Na⁺-free extracellular medium significantly reduced GHRH-stimulated GH secretion. We conclude that GHRH-induced increase in the TTX-R Na⁺ current in mouse somatotropes is mediated by the PKC system. An increase in the TTX-R Na⁺ current may contribute to the GHRH-induced exocytosis of GH granules from mouse somatotropes.

	Introduction

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GHRH PLAYS A MAJOR role in controlling GH secretion from somatotropes. It is generally accepted that GHRH depolarizes somatotropes, resulting in an increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and GH secretion. GHRH action is mediated mainly through receptor coupled Gs proteins, an increase in cAMP, and activation of protein kinase A (PKA) (1, 2, 3). The phospholipase C (PLC)-protein kinase C (PKC) signaling pathway also contributes to GH secretion (4, 5). Three main cation selective channels, Na⁺, Ca²⁺, and K⁺, have been identified in somatotropes, and their activities determine depolarization and hyperpolarization in these cells. It has been shown that GHRH increases the current through voltage-gated Ca²⁺ channels via the cAMP/PKA system. GHRH decreases the voltage-gated K⁺ current, involving the PKC system in primary cultured ovine and human somatotropes (6). It has been suggested that GHRH increases Na⁺ permeability via cAMP, which leads to depolarization of the cell (7, 8, 9) and subsequent activation of voltage-gated Ca²⁺ channels, facilitating Ca²⁺ influx and increasing [Ca²⁺]_i.

Voltage-gated Na⁺ channels have been observed in a variety of cells and are involved in regulating membrane electrical properties in excitable cells such as neurons and endocrine cells, including anterior pituitary cells (10, 11, 12, 13). Voltage-gated Na⁺ channels can be classified into two main categories: 1) those that can be selectively blocked by low, nanomolar concentrations of tetrodotoxin (TTX), called TTX-sensitive (TTX-S) Na⁺ channels and 2) TTX-resistant (TTX-R) Na⁺ channels, which are blocked only by high, tens of micromolar concentrations of TTX (14, 15). Nine subtypes of voltage-gated Na⁺ channels (Na_v1.1–1.9) have been identified and classified as a single family, based on their evolutionary relationships (14, 16). Na_v1.1–1.4 and Na_v1.6–1.7 are TTX-S, whereas Na_v1.5, -1.8, and -1.9 are TTX-R (17, 18, 19). Na_v1.5 is mainly expressed in cardiac myocytes (20, 21) and has also been reported in neuroblastoma cells (22) and rat dorsal root ganglion (DRG) neurons (23). Na_v1.8 and -1.9 are mainly expressed in DRG neurons (18, 24). Na_v1.8 mediates a slowly inactivating current and plays an important role in the generation of action potentials (25). Hyperexcitability results from an increase in the amplitude of TTX-R Na⁺ currents, and hyperexcitability is absent in DRG neurons of Na_v1.8 null mice (26). Na_v1.9 mediates an ultraslow inactivation, or persistent, TTX-R current and may contribute to modulating the resting membrane potential, hence mediating excitability (19, 27). The TTX-R Na⁺ current is well expressed and characterized in neurons but not in pituitary cells, especially somatotropes.

Kato and colleagues (7) have shown that GHRH increases [Ca²⁺]_i and GH secretion in rat somatotropes by mechanisms involving the cAMP/PKA messenger system. These effects were dependent on extracellular Na⁺ but are not blocked by 2 µM TTX (low dose), which prompted these authors to suggest the involvement of Na⁺ channels that were resistant to TTX (5, 6, 7). This notion is supported by the observation that the rise in [Ca²⁺]_i was reduced by 40% when TTX was increased to 20 µM (7). Kato (28) went on to show that GHRH increases the total voltage-gated Na⁺ current in somatotrope-enriched cells of rat pituitary. Interestingly, this current was entirely abolished by 2 µM TTX. Only seven of 16 cells responded to GHRH, and whereas this may reflect the heterogeneous nature of the cell population, it also raises the possibility that not all somatotropes express voltage-gated channels equally. To ensure that only somatotropes were investigated in the present study, we used pituitaries from green fluorescent protein (GFP)-GH transgenic mice. Thus, with new technologies, including the recent characterization of Na_v currents, we expanded the study to encompass probing the effects of PKC as well as cAMP/PKA transduction systems on TTX-R Na⁺ currents in somatotropes. We observed TTX-R Na⁺ currents using perforated whole-cell patch-clamp recordings in GFP-GH mice somatotropes. GHRH enhanced the TTX-R Na⁺ current through the PKC rather than through the PKA system.

	Materials and Methods

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Chemicals
The following solutions and chemicals were used: culture medium (DMEM), HEPES, NaHCO₃, fetal calf serum (Thermo Electron Corp., Melbourne, Australia); penicillin-streptomycin, Fungizone antibiotic solution, trypsin (Life Technologies, Inc., Gaithersburg, MD); GHRH (Auspep, Victoria, Australia); tetrodotoxin (Alomone Laboratory, Jerusalem, Israel); collagenase type I, deoxyribonuclease (DNase), hyaluronidase, trypsin inhibitor, pancreatin, tetraethylammonium Cl– (TEA), CoCl₂, nystatin, dimethyl sulfoxide (DMSO), all general salts (Sigma, St. Louis, MO); H89, KT 5720, Rp-cAMP, 8-bromoadenosine-cAMP (8-bromo-cAMP), forskolin, 3-isobutyl-1-methylxanthine (IBMX), U-73122, G6983, phorbol-12,13-dibutyrate (PDBu), and phorbol-12-myristate-13-acetate (PMA) (Calbiochem, San Diego, CA).

Cell preparation
Adult (10–12 wk) GFP-GH transgenic mice were decapitated and pituitary glands were immediately collected into ice-cold PBS solution. All experiments were approved by the Animal Ethics Committee of Monash Medical Centre (Melbourne, Australia). The pituitaries were minced and placed in Ca²⁺-free PBS with 0.1% BSA. The tissue fragments were gently washed and incubated with DNase, hyaluronidase (1 mg/ml), trypsin inhibitor (0.5 mg/ml), pancreatin, and collagenase type I (1 mg/ml) for 15 min at 37 C in a shaking bath. The solution was filtered using a sterilized mesh filter and then centrifuged at 1500 rpm for 5 min. The cells were suspended in culture medium (90% DMEM + 10% FCS + 1% antibiotic) and plated into 35-mm culture dishes for electrophysiological study and growth hormone assay. Cells were grown in a humidified incubator (37 C, 5% CO₂), the culture medium was changed every 2 d, and electrophysiological recordings were performed after 4–8 d of culture.

DNA analysis of transgenic animals
The analysis protocol has been described previously (28). Briefly, genomic DNA from tail biopsies was analyzed for transgene DNA by standard PCR procedures using PCR primers: 5'-ACC ACT CAG GGT CCT GTG GAC AG-3' (sense), 5'-CCT CTT GAA GCC AGG GCA GGC AGA GCA GGC-3' (antisense). We used a denaturation step at 94 C for 1 min, an annealing step at 60 C for 30 sec, and an extension step at 72 C for 90 sec for a total of 35 cycles, followed by an additional extension step at 72 C for 10 min. Detection of PCR amplification products was carried out by electrophoresis on a 2% agarose gel containing ethidium bromide. The signal intensity of each band was determined using Quantity One 4.5 software (Bio-Rad Laboratories, Hercules, CA).

RNA isolation and RT-PCR
The total RNA from GFP-GH mice pituitary, heart, and DRG was isolated using the RNeasy minikit fitted with a built-in column (QIAGEN, Hilden, Germany). RNA from heart and DRG were used as positive controls. Pituitary, heart, and DRG were removed immediately and frozen quickly with dry ice. RLT buffer/β-mercapto was added to the tissues and homogenized using a fine needle. The homogenate was centrifuged for 3 min on maximum (15000 rpm) speed, and then an equal volume of 70% ethanol was added to the supernatant and mixed gently by pipetting. The sample was centrifuged using an RNeasy minicolumn/collection tube for 15 sec at about 10000 rpm. RW1 buffer was added to the RNeasy minicolumn/collection tube and centrifuged for 15 sec at about 10000 rpm. Five hundred microliters of RPE buffer were added to the minicolumn and centrifuged for 15 sec at about 10000 rpm followed by addition of another 500 µl of RPE buffer and centrifuged for 2 min at about 10000 rpm. To elute the RNA, 30 µl RNA-free water was applied to the minicolumn and centrifuged for 1 min at about 10000 rpm. Total RNA extracted from each tissue was treated with DNase I (Roche, Indianapolis, IN) to eliminate possible DNA contamination. RNA (1 µg) from pituitary, heart, and DRG were then reverse transcribed to cDNA in a 20 µl reverse transcription reaction system containing random primers and avian myeloblastosis virus-reverse transcription at 46 C for 2 h.

PCR primers for Na_v1.5, -1.8, and -1.9 were designed based on published sequences of Na_v1.5 (accession no. M77235 and M27902), Na_v1.8 (accession no. X92184 and Y09108), and Na_v1.9 (accession no. NM011887) (21): for Na_v1.5, 5'-ACC AAC TGC GTG TTC ATG GCC CA-3' (sense), 5'-AGC ATG AAG AAG ATC ATG TAG ATC TT-3' (antisense) (760 bp); for Na_v1.8, 5'-GTG GAG CAC AGC TGG TTT GAG-3' (sense), 5'-AAC ACA TTC CAG CCG TTG GTG-3' (antisense) (1181 bp); for Na_v1.9, 5'-TGA TTC GCA TCT CTG TCC ATT C-3' (sense), 5'-CCC AAT GAA GAC GTA TTC GGG A-3' (antisense) (137 bp). mRNA derived from heart and DRG were used as positive controls. We used a denaturation step at 94 C for 1 min, an annealing step at 60 C for 30 sec, and an extension step at 72 C for 90 sec for a total of 35 cycles, followed by an additional extension step at 72 C for 10 min. Detection of PCR amplification products was carried out by electrophoresis on a 1.4% agarose gel containing ethidium bromide. The signal intensity of each band was determined using Quantity One 4.5 software (Bio-Rad Laboratories).

Electrophysiological recording
On the day of recording, the culture medium was replaced with patch-clamp bath solution at least 10 min before recording. Transmembrane Na⁺ currents were recorded using the patch-clamp technique in nystatin-perforated whole-cell recording (WCR) configuration. Electrodes were pulled on a P-87 microelectrode puller (Shutter Equipment Co., Navato, CA) from borosilicate glass with an inner filament (Harvard Apparatus Ltd., Edenbridge, UK). These electrodes had an initial input resistance of 4–6 M. All recording were made with the Axopatch-1 C amplifier (Axon Instruments, Foster City, CA).

The bath solution was composed of (in mM): TEA-Cl 40, NaCl 105, CaCl₂ 3, MgCl₂ 0.5, glucose 10, HEPES 10 (pH 7.4, adjusted with NaOH; osmolarity 310 mOsm/liter). To exclude contamination by Ca²⁺ currents and TTX-S Na⁺ current, 3 mM Co²⁺ and 1 µM TTX were added to the bath solution.

The pipette solution consisted of (in mM): CsCl 55, Cs₂SO₄ 75, MgSO₄ 8, and HEPES 10 (pH 7.4 and osmolarity 300 mOsm/liter), and the electrode was backfilled with this solution containing nystatin (250 µg/ml in 0.1% DMSO). This concentration of DMSO, applied to cells in 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 using a micromanipulator (Narishige, Tokyo, Japan). After obtaining a high-resistance seal the pipette potential was held at –70 mV and voltage steps (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 transient membrane capacitance 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 a greater than 20% change 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.

A gravity pressure system was used to change the bath solution. PKA-cAMP and PLC-PKC blockers/activators, including H-89, Rp-cAMP, 8-bromo cAMP, U73122, and PDBu, were added to the static bath by hand pipetting into the culture dish containing the cells. Control recordings were started at least 10 min after patching the cell. The effect of drug treatment was recorded until the response reached a plateau or until we were certain that no change was occurring. Application of vehicle did not change Na⁺ currents. All experiments were performed at room temperature (20–22 C).

Incubation experiment and mouse GH assay
After 4 d of culture in 48-well plates, each well contained approximately 30,000 cells. The cells were preincubated for 1.5 h with serum-free medium (DMEM + 0.5% BSA). This medium was subsequently discarded and 250 µl/well fresh incubation medium, with or without test substance, was applied for 45 min. The medium was collected and stored at –20 C for GH assay.

The GH concentration in the incubation medium was assayed using mouse GH-ELISA kit, DSL-10-72100 (Diagnostic Systems Laboratories Inc., Webster, TX). All samples were assayed in duplicate. All samples from one experiment were determined in the same assay, and four separate experiments were performed. GH values were expressed as nanogram equivalents of mouse GH standard.

Whole-cell current data analysis
Conductance was determined as I/(V-V_R), where I is the peak inward current at test pulse voltage V, and V_R is the calculated reversal potential. Normalized conductance, I/I_max = 1/(1 + exp[(V_pp – V_h)/k_h]), I/I_max was fit with a single Boltzmann relationship of the form where V_pp is the prepulse potential, V_h is the midpoint potential, and k_h is the slope factor. I-V curves were fitted by:

where a is a scaling factor with the dimension of conductance, V is the pulse potential, b is the potential for zero current, V_a the half-activation voltage, and k_a the corresponding slope factor.

Inactivation kinetics were evaluated by fitting the decay of the current with a single exponential for TTX-R Na⁺ current, according to the equation:

where A is amplitude, t is the interval in milliseconds, is the time constant, and C is the offset.

Data statistic analysis
The pCLAMP 8.0 software (Axon Instruments) was used to record and analyze the data. A ² test was used to normalize value percent of control, and a paired t test was used to evaluate the statistical significance of differences between control and treated group means obtained from the same group of cells. Differences were accepted as significant at P < 0.05. Group data are expressed as mean ± SEM. 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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TTX-R Na⁺ channel expression in GFP-GH transgenic mice pituitary
RT-PCR analysis showed that all three types of TTX-R Na⁺ current subtypes (Na_v1.5, -1.8, and -1.9) are expressed in cells from GFP-GH transgenic mice pituitaries. Na_v1.9 was more strongly expressed than Na_v1.5 or -1.8 (an example from one animal shown in Fig 1). As a positive control, Na_v1.5 was expressed in heart and both Na_v1.8 and -1.9 were expressed in DRG, as previously shown (18, 20). We confirmed that the PCR product did not come from DNA contamination by using RNA sample without reverse transcription (data not shown).

View larger version (29K): [in this window] [in a new window]   FIG. 1. Expression of Nav 1.5, -1.8, and -1.9 in GFP-GH transgenic mice pituitary. Ethidium bromide-stained 1.4% agarose gel showing cDNA amplified with Nav 1.5, -1.8, and -1.9 primers from RNA of GFP-GH transgenic mice pituitary, heart, and DRG. The size of amplified fragments of Nav 1.5, -1.8, and -1.9 were 760, 1181, and 137 bp, respectively.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Characterization of voltage-gated Na+ currents. A, Voltage-gated Na+ currents in response to depolarizing steps from a holding potential of –70 mV in increments of 10 mV from –50 mV to +60 mV as shown in the pulse protocols (representative traces shown). Voltage-gated Na+ currents were evoked in control solution (a), in the presence of TTX (1 µM) (b), and Na+-free solution (c). B, Current-voltage relationship of voltage-gated Na+ currents in three cells in control solution (), in the presence of TTX (), and in Na+-free solution (), measured at the peak of each current trace. Current amplitudes are normalized to the peak Na+ current for each cell. Results are mean ± SEM of three cells.

Not every GFP-positive cell possessed a TTX-R Na⁺ current. 188 of 328 GFP-positive cells, about 60% of cells, expressed a TTX-R Na⁺ current.

MrVIB (muO-conotoxin from Conus marmoreus), a blocker of Na_v1.8, had no effect on the TTX-R Na⁺ current in these somatotropes (n = 3, data not shown).

Effect of GHRH on the voltage-gated Na⁺ current in mouse somatotropes
Application of GHRH (100 nM) significantly (P < 0.05) increased the total voltage-gated Na⁺ current by about 25% (Fig. 3). All GFP-positive cells responded to GHRH. Next, the TTX-R component of the Na⁺ current was isolated using 1 µM TTX, and GHRH (100 nM) also significantly increased this TTX-R Na⁺ current, by approximately 20% (Fig. 4Ab). The effect was totally reversible within 10–15 min after removal of GHRH (Fig. 4). Mean (±SEM) values of the TTX-R Na⁺ current in five cells are shown in Fig. 4B, and increase in the Na⁺ current by GHRH was statistically significant (P < 0.05). GHRH did not affect the steady-state inactivation curve for the TTX-R Na⁺ current (Fig. 4D). Figure 4D was well fitted by a Boltzmann relationship with V_h = –60 mV (TTX-R Na⁺ current) and –61 mV (GHRH).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Voltage-gated Na+ currents in GFP-GH transgenic mice somatotropes and effect of GHRH. Voltage-gated Na+ currents were evoked from a holding potential of –70 mV. A, Data from a representative cell in control solution (a) and in the presence of GHRH (100 nM) (b). B, Mean (± SEM) Na+ current amplitude measured during depolarization steps to 0 mV (n = 5, *, P < 0.05). C, Current-voltage relationship of voltage-gated Na+ current, measured at the peak of each current trace, in the absence () and presence of GHRH (). Current amplitudes are normalized to the peak Na+ current for each cell. Results are mean ± SEM of five cells.

View larger version (15K): [in this window] [in a new window]   FIG. 4. Effect of GHRH on the TTX-R Na+ current. The voltage-gated TTX-R Na+ current in GFP-GH mice somatotropes was recorded from a holding potential of –70 mV. A, Data are from a representative cell in control solution (a), in the presence of GHRH (100 nM) (b), and after washout of GHRH (c). B, Mean (±SEM) TTX-R Na+ current amplitude measured during depolarizing steps to 0 mV (n = 5, *, P < 0.05). C, Current-voltage relationship of TTX-R Na+ current, measured at the peak of each current trace, in the absence () and presence of GHRH (), and after washout (). Current amplitudes are normalized to the peak Na+ current for each cell. Mean ± SEM of six cells. D, Steady-state inactivation curves of TTX-R Na+ current in control solution () and in the presence of GHRH (). A dual-step protocol was used in which a test step (200 msec) of 0 mV was preceded by step (100 msec) to different potentials, from –100 to –20 mV. The currents are normalized to the largest current and plotted against the prepulse voltages. Results are mean ± SEM of four cells.

The cAMP-PKA system is an important established signaling pathway in the action of GHRH in somatotropes (29, 30, 31). A series of membrane permeable blockers of cAMP-PKA signaling system was used before and during application of GHRH and the effect on the TTX-R Na⁺ current from six cells are shown in Figs. 5 and 6. The PKA inhibitor H-89 (1 µM) alone had no effect on the basal TTX-R Na⁺ current. In the presence of H-89, GHRH caused a significant (P < 0.05) increase in the TTX-R Na⁺ current that was not different from the increase observed in the absence of H89 (Fig. 5A). GHRH also evoked the usual increase in the TTX-R Na⁺ current in the presence of KT5720 (100 nM), another PKA inhibitor (Fig. 5B, P < 0.05). The competitive cAMP antagonist, Rp-cAMP (100 µM), had no effect on the basal TTX-R Na⁺ current, and it also had no effect on the ability of GHRH to significantly increase this current (Fig. 5C, P < 0.05).

View larger version (18K): [in this window] [in a new window]   FIG. 5. Effect of GHRH in the presence of cAMP-PKA blockers. Mean (± SEM, n = 6) TTX-R Na+ current amplitude in response to depolarizing steps to 0 mV from a holding potential of –70 mV. GHRH-induced an increase in the TTX-R Na+ current in the presence of PKA blockers, H-89 (1 µM) (A) and KT5270 (100 nM) (B) (*, P < 0.05). Application of GHRH in the presence of cAMP blocker, Rp-cAMP (100 µM), significantly increased TTX-R Na+ current (C) (*, P < 0.05).

View larger version (28K): [in this window] [in a new window]   FIG. 6. Effect of GHRH in the presence of cAMP-PKA activators. Mean (± SEM, n = 5 and 6) TTX-R Na+ current amplitude in response to depolarizing steps to 0 mV from a holding potential of –70 mV. A, 8-bromo cAMP (100 µM), a cAMP activator, significantly increased the TTX-R Na+ current, which was further significantly increased by GHRH (A) (*, P < 0.05). Forskolin + IBMX, AC (adenylyl cyclase) activator, increased TTX- R Na+ current significantly (B), and GHRH further increased the current (C). Conversely, stimulation with GHRH increased the Na+ current significantly, and forskolin + IBMX evoked further stimulation (C) (*, P < 0.05).

The PLC-PKC signal transduction system
Another main signaling pathway used by GHRH is the PLC-PKC system (6). Application of U73122 (5 µM), a PLC inhibitor, did not alter the basal TTX-R Na⁺ current, but it completely prevented the increase in current usually induced by GHRH (Fig. 7A). Again, GHRH failed to increase the TTX-R Na⁺ current in the presence of G6983 (1 µM), a PKC inhibitor (Fig. 7B).

View larger version (6K): [in this window] [in a new window]   FIG. 7. Effect of GHRH in the presence of PLC-PKC inhibitors. Mean (±SEM; n = 8 and 5) TTX-R Na+ current amplitude in response to depolarizing steps to 0 mV from a holding potential of –70 mV. A, U73122 (5 µM), a PLC inhibitor, had no effect on the TTX-R Na+ current but prevented the ability of GHRH to increase the current. B, Likewise, G6983 (1 µM), a PKC inhibitor, had no effect on the TTX-R Na+ current and prevented GHRH-induced stimulation.

View larger version (23K): [in this window] [in a new window]   FIG. 8. Effect of GHRH in the presence of PKC activators. Mean (±SEM, n = 4 and 6) TTX-R Na+ current amplitude in response to depolarizing steps to 0 mV from a holding potential of –70 mV. A, PDBu (0.5 µM), a PKC activator, significantly increased the TTX-R Na+ current (*, P < 0.05), but GHRH in the presence of PDBu did not increase the current. B, GHRH alone increased the TTX-R Na+ current significantly (*, P < 0.05), but application of PDBu was without additional effect. C, PMA, a PKC activator, increased the Na+ current (*, P < 0.05), but there was no further increase on addition of GHRH.

View larger version (15K): [in this window] [in a new window]   FIG. 9. Effect of GHRH in the presence of TTX and Na+-free solution on GH secretion in vitro. A, Both TTX and Na+-free solution significantly (TTX and Na+-free, P < 0.05) reduced basal GH secretion. GHRH (100 nM) increased GH secretion significantly (GHRH, P < 0.05), and this was markedly reduced by TTX (1 µM) (TTX + GHRH) and was prevented by excluding Na+ from the bathing solution (Na+ + GHRH). B, GH secretion was reformatted, expressed as percent of the appropriate control value. GHRH (100 nM) significantly increased GH secretion (*, P < 0.05) in control solution or in the presence of TTX but not in Na+-free solution.

	Discussion

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Voltage-gated Na⁺ channels are expressed in a variety of cells and they mediate the rapid increase in Na⁺ conductance during the initial phase of the action potential in many electrically excitable cells (32). The increase in the voltage-gated Na⁺ current by GHRH in rat pituitary cell has been shown previously (28), but there is no information on the specific class or type(s) of Na_v channels involved in the response to GHRH. The TTX-S current has been identified in rat somatotropes and is involved in the regulation of GH secretion (33). Indirect evidence has suggested an involvement of TTX-R Na⁺ channels in the stimulation of GH secretion by GHRH (31, 34).

Previous studies suggested that TTX-R Na⁺ channel may be involved in basal GH release by altering [Ca²⁺]_i because Na⁺-free solution immediately reduced [Ca²⁺]_i, but adding TTX did not alter basal [Ca²⁺]_i (9). In the present study, we have shown that GHRH increases the total Na⁺ current by about 25% in GFP-identified mouse somatotropes. Furthermore, by isolating the TTX-R Na⁺ current we have provided electrophysiological evidence for the existence of this component in mouse somatotropes. Here we used 1 µM TTX, which blocks all TTX-S Na⁺ channels in most tissues (15), although some suggest that cardiac TTX-S Na⁺ channels may require 10µ TTX for complete blockade (35). In the present study, 10 µM TTX did not provide additional blockade over 1 µM TTX. Na⁺ selectivity has been confirmed using choline substitution for Na⁺. The contribution of TTX-S and TTX-R channels was roughly 50% each in these somatotropes. GHRH increased the TTX-R Na⁺ current by about 20% of control, which is similar to the extent of the increase in the total Na⁺ current. It seems, therefore, that GHRH increases both TTX-S and TTX-R components of the Na⁺ current. Because the TTX-S Na⁺ current has been studied extensively in pituitary endocrine cells (36, 37, 38), the present study focused on the TTX-R Na⁺ current. GHRH did not change the kinetics of the TTX-R Na⁺ current, both the current-voltage relationship and voltage-dependent inactivation remained identical. The effect of GHRH could be explained in terms of either an increase in the number of TTX-R Na⁺ channels that open or by an increase in single channel conductance.

All three subtypes of TTX-R Na⁺ channels, Na_v 1.5, -1.8, and -1.9, are expressed in mouse pituitary gland (Fig. 1). The caveat here is that somatotropes were not isolated and hence the Na_v subtype in that cell population remains unclear. However, in the electrophysiological studies, we used a selective TTX-R Na_v1.8 channel subtype blocker, MrVIB (39), in an attempt to resolve the issue further. MrVIB had no effect on the recorded Na⁺ current. Although MrVIB is effective in blocking neuronal Na_v1.8 channels, the structure and formation of subunits of channel in somatotropes may be slightly different. Na_v1.5 channels possess fast inactivating characteristics (40, 41), whereas Na_v1.8 and -1.9 show slow or persistent inactivating characteristics, with generally longer than 4 msec (18, 19). In our somatotropes the of inactivation of the TTX-R channel was fast (1.23 ± 0.06 msec), similar to Na_v1.5 channels in other cell types. Whereas Na_v1.5 is the most sensitive of the TTX-R Na⁺ channel subtypes to TTX, being blocked in the high micromolar range (17, 23), we found that 10 µM TTX was without additional effect on the TTX-R Na⁺ current. This suggests that the TTX-R Na⁺ current in mouse somatotropes may not go through Na_v1.5. The involvement of the Na_v1.9 channel in mouse somatotropes cannot be resolved at this time and awaits future single channel recording and single cell PCR experiments.

It is generally accepted that GHRH activates the Gs protein coupled receptor signal transduction system with a consequent increase cAMP-PKA (6, 42). The PLC/PKC signaling system can interact with the cAMP/PKA pathway to effect GH secretion (4, 43). It has been shown that the voltage-gated Ca²⁺ current is increased by GHRH through the cAMP-PKA system (6). The results of the present study show that whereas the cAMP-PKA system on its own is capable of increasing the TTX-R Na⁺ current, the cAMP-PKA system is unlikely to be involved in the effects of GHRH on this current. Thus, whereas the cAMP analog, 8-bromo-cAMP, PKA inhibitors, the adenylyl cyclase activator forskolin, and the phosphodiesterase inhibitor IBMX all increased the TTX-R Na⁺ current, the cAMP antagonist, Rp-cAMP, and PKA inhibitors, H89 and KT5720, were all without effect on the increase in the TTX-R Na⁺ current evoked by GHRH.

The other important signaling pathway involved in the control of GH secretion is the PLC-PKC pathway (4, 5). Here we provide strong evidence for the involvement of the PLC-PKC system in the action of GHRH on the TTX-R Na⁺ current, which was inhibited by the PLC inhibitor, U73122, the PKC inhibitors, G6983 or chelerythrine, and after prior maximal activation of PKC by PDBu or PMA. Whereas we found that 1 µM G6983, which blocks most PKC isozymes except PKCµ, completely prevented the GHRH-induced increase in the TTX-R Na⁺ current, a lower dose of G6983 (100 nM) reduced the response to GHRH by only half. Thus, although cAMP-PKA is the main system for GH release, our result supports previous suggestions that activation of PKC is responsible for the enhancement of TTX-R Na⁺ current.

We have confirmed that GHRH (100 nM) increases GH secretion from mice somatotropes in vitro. Green fluorescent cells all responded to GHRH, and GH secretion in response to GHRH in GFP-GH mice somatotropes was similar to that in normal somatotropes (44), confirming that this as a good model. TTX reduced basal GH secretion. The augmentation of GH secretion by GHRH was completely prevented by blockade of TTX-S Na⁺ channels, indicating that TTX-S channels are crucial for GHRH-induced GH stimulation. This observation was similar to that of Kato et al. (7) using rat somatotropes. However, compared with TTX alone, GHRH still increased GH secretion in the presence of TTX. Although GHRH-induced GH secretion is less than without TTX, statistically there is no difference between GHRH and TTX + GHRH GH secretion (Fig. 9B). This indicates that the TTX-S current is not a necessary factor for GHRH-induced GH secretion. Basal GH secretion was reduced in Na⁺-free solution, but no difference was observed on basal GH secretion between TTX incubation and Na⁺-free condition (Fig. 9A). Superficially it might appear that the TTX-R Na⁺ current is not involved in basal GH release. However, the action potential in vivo likely involves both the TTX-S and TTX-R components of the Na⁺ current. Whereas half of this current can be removed using TTX, the TTX-R components cannot be manipulated selectively, thus precluding its interrogation at this stage. Thus, it may well be that halving the total Na⁺ current, by blocking either TTX-S or TTX-R, has a major impact on the strength of basic Ca²⁺ influx and hence on GH secretion. Compared with control solution or in the presence of TTX, GHRH-stimulated GH secretion in Na⁺-free solution was significantly reduced (Fig. 9B). Thus, the TTX-R Na⁺ current appears at least partially involved in the GHRH response as an essential component.

In conclusion, the TTX-R Na⁺ channel is expressed and the corresponding current has been recorded in mouse somatotropes, and the current is significantly increased by GHRH. The GHRH-induced increase in TTX-R Na⁺ current is not mediated by the cAMP-PKA signaling system but requires a normal functioning PLC-PKC transduction system. Such an increase in Na⁺ current may enhance the excitability of the cell, promoting activation of voltage-gated Ca²⁺ channels, increase Ca²⁺ influx and therefore GH secretion.

	Acknowledgments

 
We thank to Professor D. Adams for providing channel blockers and Drs. Y. Zhao, E. Vargas, and D. Keating for scientific contributions during discussion.

	Footnotes

 
This work was supported by National Health and Medical Research Council (to C.C.) and a postgraduate scholarship from the Department of Physiology, Monash University, and Prince Henry’s Institute of Medical Research (to S.-K.Y.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 5, 2008

Abbreviations: 8-Bromo-cAMP, 8-Bromoadenosine-cAMP; [Ca²⁺]_i, intracellular free Ca²⁺ concentration; DMSO, dimethyl sulfoxide; DNase, deoxyribonuclease; DRG, dorsal root ganglion; GFP, green fluorescent protein; IBMX, 3-isobutyl-1-methylxanthine; PDBu, phorbol-12,13-dibutyrate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol-12-myristate-13-acetate; TEA, tetraethylammonium; TTX, tetrodotoxin; TTX-R, TTX-resistant; TTX-S, TTX-sensitive; WCR, whole-cell recording.

Received March 24, 2008.

Accepted for publication May 28, 2008.

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