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G_i3 Mediates Somatostatin-Induced Activation of an Inwardly Rectifying K⁺ Current in Human Growth Hormone-Secreting Adenoma Cells¹

Fourth Department of Internal Medicine (K.T., J.Y.-T., T.F.), University of Tokyo School of Medicine, Tokyo 112, Japan; and Department of Neurosurgery (A.T.), Nippon Medical School, Tokyo 113, Japan

Address all correspondence and requests for reprints to: Koji Takano, M.D., Ph.D., Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3–28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan.

	Abstract

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SRIF activates an inwardly rectifying K⁺ current in human GH-secreting adenoma cells. Activation of this K⁺ current induces hyperpolarization of the membrane and abolishment of action potential firing. This mechanism is an essential mechanism for SRIF-induced decrease in intracellular Ca²⁺ concentration and inhibition of GH secretion. The activation of the inwardly rectifying K⁺ current is mediated by a pertussis toxin-sensitive G protein. In this article, the expression of the pertussis toxin-sensitive G protein -subunits in the human GH-secreting adenoma cells were analyzed by RT-PCR, and the G protein transducing the SRIF-induced activation of this inwardly rectifying K⁺ current was investigated. RT-PCR of the messenger RNA from two human GH-secreting adenomas revealed that all G_i1, G_i2, G_i3, and G_o were expressed in these adenomas. Primary cultured cells from these two adenoma cells were investigated under the voltage clamp of the whole-cell mode. Specific antibodies against the carboxyl terminus of G protein -subunits were microinjected into the cells. Microinjection of antibody against the carboxyl terminal sequence of G_i3 attenuated the SRIF-induced activation of the inwardly rectifying K⁺ current, whereas antibody against the common carboxyl terminal sequence of G_i1 and G_i2 did not. These data indicate that the G protein transducing the SRIF-induced activation of the inwardly rectifying K⁺ current is G_i3.

	Introduction

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THE INWARDLY rectifying K⁺ channels are found in a variety of tissues and cell types where they exert various functions, such as maintenance of resting membrane potential and control of cell excitability. Various kinds of hormones and regulators exert their action by regulating these channels. Many inhibitory hormones and neurotransmitters, such as SRIF and dopamine, activate these channels to inhibit the cell excitability (1).

In the pituitary, SRIF is a major physiological inhibitor of GH secretion by somatotrophs. SRIF inhibits GH secretion by inhibiting the excitability of the cell through membrane hyperpolarization. The ionic mechanism of the membrane hyperpolarization is the activation of an inwardly rectifying K⁺ current (2, 3). It is reported that a pertussis toxin-sensitive G protein mediates the activation of the inwardly rectifying K⁺ current (4). To have a precise understanding of the SRIF effect on GH secretion and the mechanism of activation of the inwardly rectifying K⁺ current, it is important to determine which G protein subtype mediates the activation of the inwardly rectifying K⁺ current. However, such information is lacking, especially in human tissue.

In this article, the expression of the pertussis toxin-sensitive G protein -subunits in the human GH-secreting adenoma cells was analyzed by RT-PCR, and the G protein subtype that mediates the coupling of SRIF receptor and the inwardly rectifying K⁺ channel was determined by using specific antibodies against the carboxyl terminal sequence of the G protein -subunits that block the coupling of the receptor and the G protein (5, 6, 7).

	Materials and Methods

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Pituitary cell culture
Written informed consent to perform in vitro research studies was obtained from all subjects. Studies were performed on tissue (pituitary adenoma) removed by transsphenoidal surgery from two patients (patient 1 and 2) with acromegaly. Preoperative serum GH levels of these patients were 59.9 ng/ml and 17.3 ng/ml in patient 1 and 2, respectively (normal < 5 ng/ml). Intramuscular injection of octreotide (100 µg) (Sandoz, Basel, Switzerland) decreased serum GH level in both patients. The lowest serum GH levels after the administration of octreotide was 4.16 ng/ml and 1.5 ng/ml, respectively. The adenoma tissues were minced into small pieces (< 1 mm) and were digested with 1000 U/ml dispase. They were seeded on 35-mm culture dishes. Cells were cultured in DMEM containing 10% heat-inactivated FCS and kept in humidified air containing 5% CO₂ at 37 C. Electrophysiological studies were performed 1–3 weeks after plating the cells.

RT-PCR
RT-PCR was used to see which of the G protein -subunits (G_i1, G_i2, G_i3, and G_o) were expressed in these GH-secreting pituitary adenomas. Messenger RNA (mRNA) was extracted from cultured GH-secreting adenoma cells using an oligo-dT beads-based method [Micro-FastTrackô kit (Invitrogen, San Diego, CA)]. TaKaRa RNA PCR kit (version 2, Takara Biomedicals, Tokyo, Japan) was used for RT-PCR. The RT solution contained 2.5 µM random 9 mers, 0.25 U/µl RT, and the extracted mRNA. mRNA from two different GH-secreting pituitary adenomas was used for analyses. Primers were designed to include intron(s), to rule out the contamination of the PCR product of the genomic DNA (8, 9). Primer sets used for PCR were 5'-ATGATCGACCGCAACCTC-3' and 5'-CTTCAACCTCCCCATAGCC-3' for G_i1, 5'-GATCGACTTTGCCGACCC-3' and 5'-TCGTTCAGGTAGTAGGCAGC-3' for G_i2, 5'-TGGCAGTGCTGAAGAAGG-3' and 5'-GGTCTTCACTCTCGTCCG-3' for G_i3, and 5'-GATGTGGTGAGTCGGATGG-3' and 5'-TGTGAAGTGGGTTTCTACGATG-3' for G_o located in the sequence common to both G_o1 and G_o2. The expected lengths of the PCR products were 227, 203, 213, and 251 bp, respectively. The PCR mixture contained 2.5 U TaKaRa Taq DNA polymerase and 0.2 µM of each primer. After a denature period of 3 min at 94 C, amplification was performed for 35 cycles at 94 C for 1 min, 52 C for 0.5 min, and 72 C for 0.5 min, by DNA thermal cycler 480 (Perkin-Elmer, Foster City, CA). The final elongation was done at 72 C for 7 min.

Direct sequencing
After confirming the expected size of the PCR products by agarose gel electrophoresis, the PCR products were purified by a PAGE. The bands of interest were cut out from the polyacrylamide gel, precipitated by isopropanol, and sequenced directly using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit and ABI 373A PRISM sequencer (Applied Biosystems, Foster City, CA).

Electrophysiology
For electrophysiological experiments, the whole-cell variation of the patch clamp technique (10) was employed. The L/M-EPC 7 amplifier (List Electronics, Darmstadt, Germany) was used to record the membrane potential or current. Application of voltage or current pulses, data acquisitions, and data analyses were conducted by using an IBM AT clone computer (Gateway, North Sioux City, ND) and a TL1–125 interface (Axon Instruments, Foster City, CA), with the pCLAMP programs (Axon Instruments). The patch electrode solution contained (in mM): 95 K aspartate, 40 KCl, 1 MgCl₂, 5 EGTA (K salt), 20 HEPES (K salt, pH 7.2), 2 ATP (Mg salt), and 0.1 GTP. The electrode resistance ranged from 5–10 M. The seal resistance was greater than several tens of G. The composition of the standard extracellular medium was (in mM): 129 NaCl, 5 KCl, 1 MgCl₂, 2.5 CaCl₂, and 10 HEPES (Na salt, pH 7.4). The liquid junction potentials were measured using glass electrodes containing 3 M KCl as a reference, and the potential values in each experiment were corrected for the liquid junction potentials (8 mV). Agents were applied to the cell by exchanging the medium, using a peristaltic pump. Experiments were carried out at room temperature (23–25 C).

Antibodies against G proteins
Two kinds of affinity-purified IgG (rabbit) against the carboxyl terminus peptides of Gi were used. One is IgG against the common carboxyl terminal sequence (345–354) of G_i1 and G_i2 (anti-G_i1/G_i2, Cat. no. 371723, Calbiochem) (2.8 mg/ml) and the other is IgG against the carboxyl terminal sequence (345–354) of G_i3 (anti-G_i3, Cat. no. 371729, Calbiochem) (4.2 mg/ml). Because the preimmune antibodies for these antibodies were not available from Calbiochem, we used nonimmune rabbit IgG (2.9 mg/ml) as the control IgG. Carboxyl terminal sequence peptide of G_i3 (345–354, antigen for anti-G_i3), to neutralize anti-G_i3, also was obtained from Calbiochem. To neutralize the antibody, 5 µl of the antigen peptide solution (50 mg/ml in 150 mM KCl solution) was added to 45 µl of the antibody solution and incubated for 1 h at 4 C. The antibody was loaded to the cell by including the antibody in the patch pipette. The antibody was diluted 200x by the internal solution. After making the whole-cell configuration, we waited for 5 min so that the antibody could diffuse into the cell. In some of the experiments, different dilutions were used.

Drugs
SRIF, ATP, and GTP were obtained from Sigma (St. Louis, MO). Dispase was obtained from Godo Shusei (Tokyo, Japan).

Statistical analyses
Statistical data were analyzed by ANOVA.

	Results

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Pertussis toxin-sensitive G proteins in GH-secreting pituitary adenoma cells
We investigated which of the pertussis toxin-sensitive G-protein -subunits (G_i1, G_i2, G_i3, and G_o) were expressed in the GH-secreting pituitary adenomas by using RT-PCR technique. mRNA from two different GH-secreting pituitary adenomas was used for analyses. Electrophoresis of the PCR products yielded a clear band at the expected size for each G protein -subunit in both adenomas. Direct sequencing of the PCR products from one of the adenomas proved that the amplified fragments using primer sets for G_i1, G_i2, G_i3, and G_o were the corresponding fragments for each G-protein -subunits, as expected. Because the primer sets for G_i1, G_i2, G_i3, and G_o were designed to contain intron(s) in between, contamination by genomic DNA was not likely. In summary, all the G_i1, G_i2, G_i3, and G_o were expressed in these GH-secreting pituitary adenomas.

G protein, transducing the SRIF-induced activation of the inwardly rectifying K⁺ current
The activation of an inwardly rectifying K⁺ current was analyzed with the conventional whole-cell clamp technique. Figure 2A shows a current record from an adenoma cell (adenoma 1) under voltage clamp at the holding potential of -48 mV. The membrane conductance was monitored by applying a 60-mV hyperpolarizing pulse step each 20 sec. Application of SRIF (10^-7 M) induced an outward shift of the holding current, accompanied by conductance increase. This outward current is the consequence of the activation of an inwardly rectifying K⁺ current, which is reported in the previous paper (3). Figure 2B shows the membrane current before (cont) and after (SRIH) the application of SRIF (10^-7 M) on a cell loaded with nonimmune affinity-purified IgG. The currents evoked by pulse step to -68 and -128 mV from the holding potential of -48 mV are shown. The current-potential relationship of the SRIF-induced current of this nonimmune IgG-loaded cell displayed distinct inward rectification (Fig. 2C). SRIF (10^-7 M) activated the inwardly rectifying K⁺ current in 8 out of 8 cells loaded with nonimmune IgG in adenoma 1, and 7 out of 7 cells in adenoma 2. Figure 3A shows the membrane currents before (cont) and after (SRIH) the application of SRIF (10^-7 M) from the cell loaded with anti-G_i1/G_i2 antibody. SRIF also activated an inwardly rectifying K⁺ current in all the cells examined (n = 8 in adenoma 1, n = 8 in adenoma 2). On the other hand, SRIF response was much attenuated in a cell loaded with anti-G_i3 antibody, as is shown in Fig. 3B (n = 11 in adenoma 1, n = 7 in adenoma 2). To see that the anti-G_i3 attenuated the response by binding to the antigen peptide, the antibody (anti-G_i3) was neutralized by mixing with the antigen peptide and incubating for 1 h at 4 C. Figure 3C shows the membrane currents obtained from a neutralized anti-G_i3-loaded cell. SRIF response was observed in the neutralized anti-G_i3-loaded cells in adenoma 1 (n = 5), indicating that the inhibition of SRIF response by anti-G_i3 was caused by the specific binding of the antibody to the antigen G protein. Figure 4, A and B, summarize the results of these experiments in adenoma 1 and 2, respectively. In the cells from adenoma 1, the effect of SRIF, expressed as SRIF-induced conductance, was 740 ± 210 pS (n = 8, mean ± SD) in cells loaded with nonimmune antibody, 800 ± 340 pS (n = 8) in cells loaded with anti-G_i1/G_i2, 52 ± 149 pS (n = 11) in cells loaded with anti-G_i3, and 650 ± 150 pS (n = 5) in cells loaded with neutralized anti-G_i3. To see whether the differences in potency of the antibody could be responsible for the different outcome between the two antibodies, we tried higher concentration for anti-G_i1/G_i2 and lower concentration for anti-G_i3. Even when the cells were loaded with 20x-diluted anti-G_i1/G_i2 (10x more concentrated than that in the experiment in Fig. 3A), the SRIF-induced conductance was not attenuated (760 ± 240 pS, n = 5). When the cells were loaded with 1000x-diluted anti-G_i3, the response still was almost abolished (73 ± 122 pS, n = 5). These indicate that the different outcome between the two antibodies may not be caused by a difference in potency. In the cells from adenoma 2, the effect of SRIF, expressed as SRIF-induced conductance, was 940 ± 280 pS (n = 7, mean ± SD) in cells loaded with nonimmune antibody, 820 ± 420 pS (n = 8) in cells loaded with anti-G_i1/G_i2, and 173 ± 94 pS (n = 7) in cells loaded with anti-G_i3. These results indicate that the activation of the inwardly rectifying K⁺ current is mediated by G_i3, and not by G_i1 or G_i2.

View larger version (12K): [in this window] [in a new window]   Figure 2. A, Membrane current record from an adenoma cell (adenoma 1) under voltage clamp at the holding potential of -48 mV. The membrane conductance was monitored by applying a-60 mV hyperpolarizing pulse step every 10 sec. Application of SRIF (10-7 M) induced an outward shift of the holding current, accompanied by a conductance increase. B, Membrane currents before (cont) and after (SRIH) the application of SRIF (10-7 M) on a cell loaded with nonimmune IgG. The dotted line indicates zero current level. The holding potential was -48 mV, and the test pulses were to -68 and -118 mV. C, I-V relationship of the SRIF-induced current. The SRIF-induced current was obtained by subtracting the membrane currents at various potentials before the application of SRIF (10-7 M) from those after the application of SRIF.

View larger version (13K): [in this window] [in a new window]   Figure 3. A, Membrane currents before (cont) and after (SRIH) the application of SRIF (10-7 M) from a cell loaded with anti-Gi1/G i2 antibody; B, membrane currents before (cont) and after (SRIF) the application of SRIF (10-7 M) from a cell loaded with anti-Gi3 antibody; C, membrane currents before (cont) and after (SRIF) the application of SRIF (10-7 M) from a cell loaded with neutralized anti-Gi3 antibody. Dotted lines indicate zero-current level. The holding potential was -48 mV and the test pulses were to -68 and -118 mV.

View larger version (23K): [in this window] [in a new window]   Figure 4. Summary of the effect of SRIF (10-7 M) on the membrane current from cells loaded with various antibodies. A, The increase in slope conductance (% of the control) in each antibody-injected cell from adenoma 1; cont, data from cells loaded with nonimmune IgG; anti-Gi1/i2, data from cells loaded with anti-Gi1/Gi2; anti-Gi3, data from cells loaded with anti-Gi3; neutr. anti-Gi3, data from cells loaded with neutralized anti- Gi3; B, the increase in slope conductance (% of the control) in each antibody-injected cell from adenoma 2; cont, data from cells loaded with nonimmune IgG; anti-Gi1/i2, data from cells loaded with anti-Gi1/Gi2; anti-Gi3, data from cells loaded with anti-Gi3. Data were analyzed by ANOVA; *, P < 0.05. The bar indicates 1 SD.

	Discussion

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The G protein subtype that couples the SRIF receptor to the inwardly rectifying K⁺ channel has not been determined yet. To solve this problem clearly, it is mandatory to use a method that can block the transduction pathway in a well-characterized manner. For this purpose, we loaded the cells with antibodies against the carboxyl termini of the G protein -subunits that block the receptor-G protein coupling (5, 6, 7). By this method, we determined that G_i3 mediates the activation of the inwardly rectifying K⁺ current in GH-secreting adenoma cells. The anti-G_i3 we used in the experiment was specific and did not cross-react to other G-subtypes. However, the antibody against the carboxyl terminus of G_o protein available for us, cross-reacted to G_i3, probably because the carboxyl terminal sequences of these two G protein -subunits are identical. Therefore, we did not perform the experiments using this anti-G_o/G_i3, which cannot discriminate the two G protein -subunits. However, the finding that anti-G_i3 almost abolished the SRIF response indicates that the involvement of G_o in the SRIF response may not be a major one.

We performed experiments to reveal which G protein -subunits are found in these cells. Because the amounts of the adenoma tissues were very limited, it was not possoble to perform Southern blotting or Northern blotting to evaluate the precise amount of each G-subunit. Instead, we used RT-PCR to examine the expression of each G protein -subunit. We amplified the four pertussis toxin-sensitive G protein -subunits by RT-PCR, using specific primer sets for each G, and found that all four subunits are expressed in both of these tumors. Therefore, the finding that anti-G_i3 alone abolished the response was not caused by the absence of G_i1 or G_i2 in the cells. The amount of the RT-PCR product for G_i3 was not larger than those of other G protein -subunits.

Interestingly, G_i3 is known to transduce the activation of a delayed rectifier K⁺ current by dopamine through D₂ receptor in PRL-secreting rat anterior pituitary cells (19, 20). The fact that activation of the inwardly rectifying K⁺ channels and delayed rectifier K⁺ channels by inhibitory hormone was, in both cases, mediated by G_i3 suggests a unique tertiary conformation of G_i3 for coupling to K⁺ channels.

Because growing evidence is indicating that activation of inwardly rectifying K⁺ channels by pertussis toxin-sensitive G proteins is mediated by ß dimers, rather than -subunit (17, 18, 21), our findings that antibody to a single -subunit (anti-G_i3) almost abolished the SRIF-induced activation of the inwardly rectifying K⁺ current is somewhat surprising. In atrial cells, different types of ß-subunits are capable of activating inwardly rectifying K⁺ channels (21). In locus ceruleus neurons, G protein ß-subunits activate the inwardly rectifying K⁺ channel directly (22). Because more than one G protein (G_i1, G_o, and G_i3) couple to SRIF receptors (23, 24, 25) and different types of ß-subunits are capable of activating the inwardly rectifying K⁺ channel, why does the ß-subunit (which couples to the G_i3) activates only the inwardly rectifying K⁺ current, not the ß-subunits (which couple to other G protein -subunits)? Recently, a theory that could answer this question was presented by investigators (26, 27). Their data suggested that the three components (receptor, G protein, and G protein-activated inwardly rectifying K⁺ channel) form a complex under physiological conditions. Because of the physical proximity of these components, only a certain type of G protein can activate the inwardly rectifying K⁺ current. According to their theory, the specificity of the G protein observed in our experiments resides in the coupling of the G protein -subunit and the SRIF receptor, and the ß-subunit associated with the G_i3-subunit in the receptor-G protein-channel complex specifically activates the inwardly rectifying K⁺ current because of the physical proximity.

Inwardly rectifying K⁺ current plays an essential role in the mechanism of SRIF-induced inhibition of GH secretion. Activation of this current by SRIF hyperpolarizes the membrane and abolishes the action potential firing (2). Inhibition of action potential firing reduces the Ca²⁺ influx through the voltage-gated Ca²⁺ channel and thus decreases intracellular Ca²⁺ concentration, which is a major regulator of GH secretion. Therefore the assignment of the G protein subtype transducing the SRIF-induced activation of the inwardly rectifying K⁺ current in human somatotroph is very important, not only in the investigation of human physiology of GH secretion, but also in understanding the mechanism of action of SRIF-analogue that is used for the medical treatment of diseases of abnormal GH secretion, such as gigantism and acromegaly. This result also is important for understanding the mechanism of the G protein-effector coupling.

View larger version (56K): [in this window] [in a new window]   Figure 1. RT-PCR detection of G mRNA from two different GH-secreting pituitary adenomas. The PCR products, amplified by corresponding set of primers, were analyzed by agarose gel electrophoresis. The amplified G-subunits are Gi1 (lanes 2 and 6), Gi2 (lanes 3 and 7), Gi3 (lanes 4 and 8), and Go (lanes 5 and 9). No bands were detected in the PCR controls (no complementary DNA) (data not shown). Lane 1, 100-bp DNA ladder.

	Acknowledgments

	Footnotes

 
¹ This work was supported by grants from the Ministry of Education (Japan).

Received October 28, 1996.

	References

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