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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.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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
Gi1, Gi2, Gi3, and
Go 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
Gi3 attenuated the SRIF-induced activation of the
inwardly rectifying K+ current, whereas antibody against
the common carboxyl terminal sequence of Gi1 and
Gi2 did not. These data indicate that the G protein
transducing the SRIF-induced activation of the inwardly rectifying
K+ current is Gi3. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR
RT-PCR was used to see which of the G protein -subunits
(Gi1, Gi2, Gi3, and
Go) 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
Gi1, 5'-GATCGACTTTGCCGACCC-3' and
5'-TCGTTCAGGTAGTAGGCAGC-3' for Gi2,
5'-TGGCAGTGCTGAAGAAGG-3' and 5'-GGTCTTCACTCTCGTCCG-3' for
Gi3, and 5'-GATGTGGTGAGTCGGATGG-3' and
5'-TGTGAAGTGGGTTTCTACGATG-3' for Go located in the
sequence common to both Go1 and Go2. 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 Gi1 and
Gi2 (anti-Gi1/Gi2, Cat.
no. 371723, Calbiochem) (2.8 mg/ml) and the other is IgG against the
carboxyl terminal sequence (345–354) of Gi3
(anti-Gi3, 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
Gi3 (345–354, antigen for anti-Gi3), to
neutralize anti-Gi3, 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits (Gi1, Gi2,
Gi3, and Go) 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 Gi1,
Gi2, Gi3, and Go were the
corresponding fragments for each G-protein -subunits, as expected.
Because the primer sets for Gi1, Gi2,
Gi3, and Go were designed to contain
intron(s) in between, contamination by genomic DNA was not likely. In
summary, all the Gi1, Gi2,
Gi3, and Go 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-Gi1/Gi2
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-Gi3 antibody, as is shown in
Fig. 3B (n = 11 in adenoma 1, n = 7 in adenoma 2). To see
that the anti-Gi3 attenuated the response by binding to
the antigen peptide, the antibody (anti-Gi3) 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-Gi3-loaded cell. SRIF response was
observed in the neutralized anti-Gi3-loaded cells in
adenoma 1 (n = 5), indicating that the inhibition of SRIF response
by anti-Gi3 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-Gi1/Gi2,
52 ± 149 pS (n = 11) in cells loaded with
anti-Gi3, and 650 ± 150 pS (n = 5) in cells
loaded with neutralized anti-Gi3. 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-Gi1/Gi2 and lower
concentration for anti-Gi3. Even when the cells were
loaded with 20x-diluted anti-Gi1/Gi2
(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-Gi3, 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-Gi1/Gi2, and 173 ± 94 pS
(n = 7) in cells loaded with anti-Gi3. These
results indicate that the activation of the inwardly rectifying
K+ current is mediated by Gi3, and not by
Gi1 or Gi2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit released from the trimeric G protein complex activates
the channel directly (14). However the G protein subtype transducing
the SRIF signal has not been determined. Yatani and others (15) have
shown that antibody against Gk (a hypothetical G protein
that transduces the inhibitory neurotransmitter-induced activation of
the K+ channel) inhibited the muscarine-induced activation
of the single-channel activities. They also have shown that -subunit
of purified Gk (k), which was identical to
Gi3, activates the same K+ channel that is
activated by SRIF and carbachol (16). However, accumulating evidence is
indicating that activation of inwardly rectifying K+
channels by pertussis toxin-sensitive G proteins is mediated by ß
dimers, rather than -subunit (14, 17, 18). Therefore, the results by
Yatani and others, indicating that Gi3-subunit directly
activates the SRIF-activated K+ channels, need
reevaluation.
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 Gi3
mediates the activation of the inwardly rectifying K+
current in GH-secreting adenoma cells. The anti-Gi3 we
used in the experiment was specific and did not cross-react to other
G-subtypes. However, the antibody against the carboxyl terminus of
Go protein available for us, cross-reacted to
Gi3, probably because the carboxyl terminal sequences of
these two G protein -subunits are identical. Therefore, we did not
perform the experiments using this
anti-Go/Gi3, which cannot discriminate
the two G protein -subunits. However, the finding that
anti-Gi3 almost abolished the SRIF response indicates
that the involvement of Go 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-Gi3 alone abolished the
response was not caused by the absence of Gi1 or
Gi2 in the cells. The amount of the RT-PCR product for
Gi3 was not larger than those of other G protein
-subunits.
Interestingly, Gi3 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 Gi3 suggests a unique tertiary conformation of Gi3 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-Gi3) 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 (Gi1, Go, and
Gi3) 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 Gi3) 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 Gi3-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.
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Acknowledgments |
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Footnotes |
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Received October 28, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or ß subunits. Methods
Enzymol 195:215–233[Medline]
subunit. J Biol Chem 263:6656–6664 chain of GTP-binding regulatory protein Go: two
different mRNAs are generated by alternate splicing. Proc Natl Acad Sci
USA 88:2974–2978 subunit of GTP-binding proteins activate the
muscarinic K+ channel in heart. Nature 325:321–326[CrossRef][Medline]
3 mediates DA inhibition of PRL release
while masking its PRL-releasing activity. Endocrinology 135:790–793[Abstract]
-subunits activate the muscarinic-gated atrial
potassium channel. Nature 368:255–257[CrossRef][Medline]
subunits with cell
membrane. FEBS Lett 390:217–220[CrossRef][Medline]
1
selectively couples somatostatin receptors to adenylyl cyclase in
pituitary-derived AtT-20 cells. Mol Pharmacol 41:452–455[Abstract]
to the GIRK1 G
protein-gated inwardly rectifying K+ channels is important
for channel activation. Neuron 15:1133–1143[CrossRef][Medline]
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