Gi-3 protein mediates the increase in voltage-gated K+ currents by somatostatin on cultured ovine somatotrophs

Chen Chen

Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia

    ABSTRACT
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Abstract
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Materials & Methods
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Voltage-gated K+ currents in rat somatotrophs are increased by somatostatin (SRIF) through unidentified G protein. In this experiment, somatotroph-enriched cells (up to 85%) were obtained from ovine pituitary glands and further identified by the increase in K+ currents by SRIF. The whole cell recording was employed to study the voltage-gated K+ currents. A reversible increase in K+ currents (up to 150% of control) was obtained in response to local application of SRIF (10 nM) but not vehicle. When the guanosine 5'-O-(3-thiotriphosphate) was included in the pipette solution (200 µM), the recovery phase of K+ current response to SRIF was abolished. Inclusion of guanosine 5'-O-(2-thiodiphosphate) (200 µM) in pipette solution blocked the K+ current response to SRIF. Intracellular dialysis of antibodies against alpha o-, alpha i-, alpha i-1-2-, or alpha i-3-subunits of G proteins via patch pipettes was confirmed by immunofluorescent staining of the antibodies. Antibody dialysis alone did not modify voltage-gated K+ currents. Dialysis of anti-alpha i or anti-alpha i-3 antibodies significantly attenuated the increase in K+ currents that was obtained after application of 10 or 100 nM SRIF. Dialysis with anti-alpha o, anti-alpha i-1-2, or heat-inactivated (60°C for 10 min) anti-alpha i antibodies did not diminish the effect of SRIF on K+ currents. We conclude that the Gi-3 protein mediates the effect of SRIF on voltage-gated K+ currents in ovine somatotrophs.

pituitary; growth hormone; patch clamp; signaling; antibodies; dialysis

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A NUMBER of different ion channels in the somatotroph cell membrane are modified by somatostatin (SRIF), leading to a reduction in growth hormone (GH) secretion (7). The direct mechanism for such a reduction in GH secretion is a decrease in intracellular free Ca2+ concentration ([Ca2+]i). Such an effect can be achieved by a decrease in transmembrane Ca2+ currents, as has been observed in pituitary somatotrophs (4, 8, 20) and neurons (15, 25). The reduction in Ca2+ influx can also be reached by an indirect mechanism through the action of SRIF on K+ channels. It has been shown, in rat somatotrophs, that SRIF increased several types of K+ currents including voltage-gated transient (IA) and delayed rectifying (IK) currents (6, 9) as well as inwardly rectifying K+ currents (24).

The SRIF receptor has a structure that typically couples to G proteins (1). It has been suggested that G proteins mediate the effect of SRIF on Ca2+ currents (4, 8, 15, 25). The effect of SRIF on K+ currents has also been demonstrated to be an action coupled by G protein (18, 19). Subtypes of the SRIF receptor are thought to be coupled to different types of G protein (1), and the Gi protein is believed to mediate the effect of SRIF on K+ channels (32) in GH tumor cells. It has recently been demonstrated that Go-2 protein mediates the effect of SRIF on membrane Ca2+ currents in "normal" ovine somatotrophs in primary culture (4); this protein may also play a role in the effect of SRIF to reduce Ca2+ currents in GH3 tumor cells (17). The subtype of G protein mediating the augmentation of K+ currents by SRIF is, however, not clear. The present study aimed to resolve this issue by dialysis of antibodies against the alpha -subunit of G proteins into cells to functionally block the action of certain subtypes of G proteins. In cultured ovine somatotrophs, we found that the alpha i-3-subunit of Gi protein (Gi-3) mediates the increase in the voltage-gated K+ currents by SRIF. The preliminary data have previously been reported at the Australian Physiological and Pharmacological Society Symposium (3).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell preparation. Sheep pituitaries were obtained from a local abattoir and then subjected to collagenase-pancreatin treatment to liberate cells as described previously (5). Cell yield was usually >107 cells/pituitary gland, with >90% viability (trypan blue exclusion test). The cell suspension (3-5 ml) was placed, under sterile conditions, above a layered column of Percoll solutions of increasing density and centrifuged as previously described (5). Cells in fractions 1 and 2 contained up to 85% of somatotrophs and were plated onto 35-mm petri dishes and used in these experiments (5). Electrophysiological recordings were made after 4-14 days in culture in a humidified incubator (37°C, air 95%-CO2 5%). In each case penicillin-streptomycin was used in the culture for the first 24 h in vitro. The culture medium was then changed every 48 h by using DMEM plus 10% sheep serum and 2% FCS.

Cell dialysis and experimental design. Cell dialysis via patch pipette of molecules of various molecular weights was carefully studied by Pusch and Neher (21). From their equations and our previous experience (4), ~72% of the antibody concentration in the patch pipette was dialyzed into the cell within 25 min with an electrode of resistance of 5 MOmega . We therefore used a dialysis time of 25 min to allow adequate transfer of the antibodies into the cell.

Specific antibodies to alpha o-, alpha i-, alpha i-1-2-, or alpha i-3-subunits were used in the electrode solution at a concentration of 1:100-200 in presence of BSA (0.5%). Anti-alpha o-antibody (GC/2) specifically against alpha o-subunit without cross-reactivity to alpha i-1-, alpha i-2-, alpha i-3-, or alpha s-subunits was supplied by Du Pont. Anti-alpha i-1-2-antibody (AS/7) without cross-reactivity to alpha o-, alpha s-, or alpha i-3-subunits was also supplied by Du Pont. Anti-alpha i-1-3-antibody (C-10) without cross-reactivity to alpha o- or alpha s-subunits was supplied by Santa Cruz Biotechnology. Anti-alpha i-3-antibody (to COOH-terminal 345-354) without cross-reactivity to alpha i-1-, alpha i-2-, alpha o-, or alpha s-subunits was supplied by Calbiochem. Transmembrane K+ currents were recorded by depolarizing the membrane potential from a holding potential of -80 mV to +40 or +20 mV for 200 ms. Depolarizing pulses were applied at 1-min intervals. The first application of SRIF was made within 5 min of establishing whole cell recording (WCR), and a second application was made after 25 min (i.e., 25 min after antibody dialysis).

Immunocytochemistry. As described in our previous experiments, an immunofluorescent staining technique was used to verify the dialysis of antibodies into the cell (4). After electrophysiological recordings with electrode solution containing antibodies, recorded cells were washed in PBS and fixed in 4% paraformaldehyde for 15-30 min. The cells were then incubated in 50:50 acetone-methanol for 3 min, washed with PBS, and then incubated for 30 min in PBS containing 5% horse serum to eliminate nonspecific binding. After further washing with PBS, the cells were exposed to a 1:30 (as suggested by the producer) dilution of FITC-conjugated anti-rabbit IgG antibodies for 1 h at room temperature and then mounted under coverslips in 70% glycerol for storage at 4°C before viewing. Negative control was established on the dishes without antibody dialysis and no cells stained positive. Only cells dialyzed with rabbit antibodies stained positive.

Electrophysiological recording. Transmembrane currents were recorded by using the "gigaseal" patch-clamp technique in WCR configuration (14), and the peak value of each current trace was reported here. All recordings were made with the Axopatch 200 amplifier, and electrodes were pulled by a Sutter P-87 microelectrode puller from borosilicate micropipettes coated with wax and fire polished. The tip resistance of the electrode filled with internal solution ranged from 2 to 5 MOmega . Recordings were made on the stage of an Olympus inverted microscope. The bath solution was composed of the following: 140 mM NaCl, 0.5 mM CaCl2, 1 mM CdCl2, 5 mM KCl, 0.5 mM MgCl2, 10 mM HEPES, 10 mM glucose, and 1 µM tetrodotoxin at pH 7.4 and osmolarity of 310 mosM. The electrode solution was composed of the following: 140 mM KCl, 10 mM EGTA, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose. An ATP regenerative system (2 mM ATP, 5 mM phosphocreatine disodium salt, and 20 U/ml creatine kinase) and the antibodies were added into the electrode solution just before recording, and the electrode solution was adjusted to pH 7.4 and osmolarity of 300 mosM.

Original cell culture dishes were fixed on the stage of the microscope, and a peristaltic pump was used to perfuse the cells at a rate of 1 ml/min. Short-term SRIF application was performed by using gravity flow through a large-bore pipette (~10 µM) located ~0.5 cm from the recorded cell. Injection with vehicle did not cause any change in K+ currents. Long-term SRIF treatment was achieved by changing the perfusion medium.

Data analysis and chemicals. Figures showing K+ current data represent one example from a group of experiments. The data are presented as means ± SE calculated from at least six experiments. Effects of treatments were considered significant at P < 0.05 level by ANOVA.

Culture media were obtained from Cytosystems (Castle Hill, NSW, Australia), sera and pancreatin were from GIBCO (Gaithersburg, MD), collagenase was obtained from Worthington Biochemical (Freehold, NJ), and SRIF was purchased from Auspep (Parkville, VIC, Australia). Antibodies to alpha o- and alpha i-1-2-subunits of G proteins (GC/2 and AS/7, respectively) were purchased from Du Pont-Australia (North Ryde, NSW, Australia). Antibodies to alpha i-3-subunit of G protein were obtained from Calbiochem (catalog no. 371729; San Diego, CA), and antibodies to alpha i-1-3-subunit of G protein were from Santa Cruz Biotechnology (catalog no. sc-262; Santa Cruz, CA). DNase and all salts for experimental solutions were purchased from Sigma (St Louis, MO).

    RESULTS
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Abstract
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Materials & Methods
Results
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Evidence for successful dialysis of antibodies into recorded cells. The dialysis of antibodies into cells was verified by immunofluorescent staining with FITC-conjugated anti-rabbit IgG antibodies. The staining of the recorded cells indicates that the antibodies were successfully and specifically dialyzed into the cell during the WCR (Fig. 1). Similar results were obtained for all cells in this experiment with dialysis of antibodies.


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Fig. 1.   Immunofluorescent staining of anti-alpha -subunit antibodies. Identification of antibodies in a cell dialyzed by anti-alpha i-3-subunit antibodies raised from rabbit. A: recorded cell under light microscopy. B: fluorescent staining of this cell with FITC-conjugated anti-rabbit IgG antibodies. Note that other cells in field were not stained by anti-rabbit IgG antibodies, indicating specificity of staining for dialyzed antibody.

Effect of guanosine 5'-O-(3-thiotriphosphate) or guanosine 5'-O-(2-thiodiphosphate) on voltage-gated K+ current response to SRIF. It has been shown that SRIF causes an increase in voltage-gated K+ currents in rat somatotrophs (9). In ovine somatotrophs, two types of voltage-gated K+ currents were recorded as transient, 4-aminopyridine-sensitive IA current and delayed rectifying, tetraethylammonium-sensitive IK current, with the IK being dominant (5). The peak voltage-gated K+ current (combined IA and IK) was subsequently investigated in this experiment to simplify the data. Local administration of SRIF increased both IA and IK currents without preference in this experiment in agreement with our previous observation in rat somatotrophs (9). Such an increase in the voltage-gated K+ currents induced by 10 nM SRIF was reversible (Fig. 2, A and C) after removal of SRIF. When guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S, 200 µM) was included in the electrode solution, application of SRIF increased K+ currents, but this increase was not reversible after removal of SRIF (Fig. 2, B and D). When guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S, 200 µM) was included in the electrode solution, the first application of SRIF induced a reversible increase in the voltage-gated K+ currents that was the same as the control group (Fig. 3, A and C). After exposure to SRIF one or two times in the presence of GDPbeta S in the electrode, the increase in the K+ currents induced by SRIF was totally abolished, whereas the cells in the control group responded to SRIF in a manner compatible with that seen after the first application of SRIF (Fig. 3, B and C). This is probably due to an increase in diffusion of GDPbeta S to the area close to G protein and/or the competition of GDPbeta S with GTP in the cell for the replacement of GDP binding of alpha -subunits during response to SRIF. Dialysis of GDPbeta S did not affect the kinetics or amplitude of the K+ currents. These effects of GTPgamma S and GDPbeta S dialysis strongly confirmed the involvement of G proteins in the voltage-gated K+ current response to SRIF.


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Fig. 2.   Effects of intracellular dialysis of guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) on K+ current response to somatostatin (SRIF). A: K+ current was evoked by depolarizing pulses from a holding potential of -80 to +40 mV as indicated at bottom, with a normal electrode solution. Traces: control, control current; SRIF, current after application of 10 nM SRIF; removal of SRIF, current 10 min after removal of SRIF. B: K+ current was evoked by depolarizing pulses from a holding potential of -80 to +40 mV as indicated at bottom, with an electrode solution containing GTPgamma S (200 µM). Traces as in A. C: K+ current (calculated for unit membrane capacitance, pF) was evoked by depolarizing pulses (1 pulse/min) from a holding potential of -80 to +40 mV with standard K+ electrode solution. Data represent experiment shown in A. D: K+ current (calculated for unit membrane capacitance, pF) was evoked by depolarizing pulses (1 pulse/min) from a holding potential of -80 to +40 mV with standard K+ electrode solution containing GTPgamma S for intracellular dialysis. Data represent experiment shown in B.


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Fig. 3.   Effects of intracellular dialysis of guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S) on K+ current response to SRIF. A: K+ current was evoked by depolarizing pulses from a holding potential of -80 to +40 mV as indicated at bottom, with an electrode solution containing GDPbeta S (200 µM). Traces as in Fig. 2 legend except as follows: SRIF, current after 1st application of 10 nM SRIF. B: K+ current was evoked by same protocol in same cells as in A. Traces: SRIF, current after 3rd application of 10 nM SRIF; removal of SRIF, current 10 min after removal of 3rd application of SRIF. C: K+ current (calculated for unit membrane capacitance, pF) was recorded by depolarizing pulses (1 pulse/min) from a holding potential of -80 to +40 mV with standard K+ electrode solution (open columns) or an electrode solution containing GDPbeta S (200 µM, filled columns) for intracellular dialysis. Data represent mean ± SE (n = 9) peak K+ current and K+ current in presence of 10 nM SRIF during 1st application of SRIF (5 min) or 3rd application of SRIF >25 min after whole cell recording (WCR; 25 min) was established. * P < 0.05.

Dialysis of antibodies on K+ current response to SRIF. Dialysis of antibodies did not affect the kinetics of the voltage-gated K+ current (see below). With WCR using anti-alpha i-1-3 antibodies in the pipette, the increase in K+ current by SRIF (10 nM) was obtained within 5 min of establishing WCR (Fig. 4B, traces at top). This response diminished when SRIF was given a second time after 25 min of dialysis with anti-alpha i-1-3 antibodies (Fig. 4B, traces at bottom). With anti-alpha o-antibodies in the electrode solution, the increase in K+ current by SRIF (10 nM) after 25 min of dialysis was similar to the control responses (Fig. 4A). Dialysis with heat-inactivated (60°C for 10 min) anti-alpha i-1-3-antibodies did not affect the K+ current response to SRIF after 25 min of dialysis (results not shown). Dialysis of anti-alpha o- or anti-alpha i-1-3-antibodies did not change the basal voltage-gated K+ current recorded by depolarizing the membrane potential from a holding potential of -80 to +20 mV (Fig. 4, A and B). The change in K+ current density (K+ current/unit capacitance) from a group of eight cells is shown in Fig. 4C, indicating that anti-alpha i-1-3-antibodies significantly (P < 0.01) decreased the K+ current response to SRIF. This dialysis of anti-alpha i-1-3-antibodies blocked the response to SRIF even when a very high dose of SRIF (10-6 M) was used (6.5% of initial response, n = 5; data not shown). When anti-alpha o-antibodies were included in electrode solution, the modification of the SRIF response did not occur (Fig. 4C).


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Fig. 4.   Effect of intracellular dialysis of anti-alpha o or anti-alpha i-1-3-subunits of G protein antibodies on SRIF-induced increase in K+ currents. A: K+ currents were evoked by depolarizing pulses from a holding potential of -80 to +20 mV as indicated at bottom, with an electrode solution containing anti-alpha o-antibodies. SRIF was applied twice within 5 min and again at 25 min after WCR was established. Traces as in Fig. 2 legend here and in B. B: K+ currents were evoked by depolarizing pulses from a holding potential of -80 to +20 mV as indicated at bottom, with an electrode solution containing anti-alpha i-1-3-antibodies. SRIF was applied twice within 5 min and again at 25 min after WCR was established. C: K+ current (calculated for unit membrane capacitance, pF) was evoked by depolarizing pulses from a holding potential of -80 to +20 mV with an electrode solution containing antibodies against alpha o- or alpha i-1-3-subunit of G protein for intracellular dialysis. Data represent mean ± SE peak K+ current (control) and K+ current in presence of 10 nM SRIF during 1st application of SRIF (5 min) or 25 min after WCR was established (25 min) during intracellular dialysis with anti-alpha o- and anti-alpha i-1-3-antibodies (n = 8). ** P < 0.01.

When anti-alpha i-3-antibodies were included in the electrode solution, the K+ current response to SRIF was significantly reduced after 25 min of dialysis (Fig. 5, B and C), whereas anti-alpha i-1-2-antibodies did not modify the K+ current response to SRIF (Fig. 5, A and C).


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Fig. 5.   Effect of intracellular dialysis of anti-alpha i-1-2- or anti-alpha i-3-subunits of G protein antibodies on SRIF-induced increase in K+ current. A: K+ current was evoked by depolarizing pulses from a holding potential of -80 to +40 mV as indicated at bottom, with an electrode solution containing anti-alpha i-1-2-antibodies. SRIF was applied twice within 5 min and again at 25 min after WCR was established. Traces as in Fig. 2 legend here and in B. B: K+ current was evoked by depolarizing pulses from a holding potential of -80 to +40 mV as indicated at bottom, with an electrode solution containing anti-alpha i-3-antibodies. SRIF was applied twice within 5 min and again at 25 min after WCR was established. C: K+ current (calculated for unit membrane capacitance, pF) was evoked by depolarizing pulses from a holding potential of -80 to +40 mV with an electrode solution containing antibodies against alpha i-1-2- or alpha i-3-subunit of G protein for intracellular dialysis. Data represent mean ± SE peak K+ current (control) and K+ current in presence of 10 nM SRIF during 1st application of SRIF (5 min) or 25 min after WCR was established (25 min) during intracellular dialysis with anti-alpha i-1-2- and anti-alpha i-3-antibodies (n = 5 and 8, respectively). ** P < 0.01.

    DISCUSSION
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This experiment provides convincing evidence that the action of SRIF on voltage-gated K+ currents in ovine somatotrophs is mediated by Gi-3 protein. Structurally, the receptors for SRIF on somatotrophs are of the type that contains seven transmembrane domains, suggesting that they couple to G proteins (1). Several lines of evidence in support of this notion use various approaches, such as direct application of purified Gialpha protein in inside-out patch-clamp recording (32), pretreatment of cells with pertussis toxin, which abolishes the function of certain types of G proteins (27), or intracellular application of GTPgamma S to abolish the deactivation of G proteins after the activation by SRIF (13, 27). The types of K+ channels that may be modified by SRIF in various tissues include inwardly rectifying K+ channels (Kir), ATP-sensitive K+ channels (KATP), Ca2+-activated K+ channels, and voltage-gated K+ channels (2, 9, 28, 30). In the present experiment, we showed that the increase in voltage-gated K+ currents by SRIF became irreversible after intracellular dialysis with GTPgamma S and were abolished after dialysis with GDPbeta S. These data strongly suggest that the action of SRIF on voltage-gated K+ channels is mediated by G proteins.

This finding was extended to the subtypes of G proteins by using antibody dialysis techniques recently established in our laboratory (4). Dialysis of antibodies against the alpha i-3-subunit significantly attenuated the SRIF-induced augmentation of the voltage-gated K+ currents, whereas antibodies against the alpha i-1-2- or alpha o-subunits did not. This therefore indicates that the G protein transducing the SRIF-induced increase in voltage-gated K+ currents in ovine somatotrophs is Gi-3 protein. In human GH tumor cells, it has recently been reported that the same type of G protein mediates the action of SRIF on Kir currents using similar techniques (microinjection of antibodies) (28). This suggests that a similar signaling system be used by SRIF to increase both voltage-gated K+ currents and Kir currents. Because of the ubiquitous distribution of multiple types of G proteins, detailed study of the subtypes of G proteins at single cell level is required to resolve which subunits mediate particular responses. Intracellular dialysis of antibodies and/or antisense oligonucleotides via patch-clamp electrode provides a powerful approach in this regard (4).

In ovine somatotrophs, we previously demonstrated that modulation of voltage-gated K+ currents altered the membrane resting potential as well as the shape and timing of action potentials (5). Increases in these K+ currents by SRIF will contribute to the inhibition of GH secretion by SRIF via a reduction in Ca2+ influx through voltage-gated Ca2+ channels (5, 9). Existing evidence also indicates that the reduction in voltage-gated Ca2+ currents induced by SRIF is partially responsible for the decrease in Ca2+ influx (4, 8, 20, 29). Other types of K+ currents, such as Kir and KATP, play pivotal roles in maintenance of the resting membrane potential and are enhanced by SRIF in pituitary or pancreatic beta -cells (2, 16, 28). We did not observe significant Kir or KATP currents in ovine somatotrophs (5); this may reflect a species difference or a difference in experimental conditions. Ca2+-activated K+ channels have recently been reported to regulate the release of prolactin in rat pituitary cells (30) but may not be involved in the response to SRIF in somatotrophs. It is likely that the inhibitory effect of SRIF on GH secretion is achieved by its action on multiple types of SRIF receptors in pituitary somatotrophs to modify multiple types of ion channels on the cell membrane.

The second messenger systems responsible for the increase in K+ currents is not yet clear. The alpha i-subunit of Gi protein mediates the inhibitory effect of SRIF on cAMP production in neurons (31). There is evidence, however, to suggest an uncoupling between cAMP and the effect of SRIF on K+ channels in human or rat pituitary adenoma cells (10, 26). Phosphorylation of K+ channel proteins has been linked to a change of K+ conductance of Ca2+-activated K+ channels (11), probably through protein kinase (PK) C activation (23). It is still not clear whether such a system is involved in the action of SRIF on voltage-gated K+ channels. It has been shown in GH4 cells that a cell-permeable cAMP analog reduced the voltage-gated K+ currents and this was completely reversed by SRIF (10). This indicates that SRIF may not act on voltage-gated K+ channels via the reduction in cAMP levels or subsequent reduction in PKA activity. In addition, the action of SRIF on voltage-gated K+ channels was recorded in classical whole cell patch-clamp configuration. Cytoplasm second messenger systems may have been washed out during recording; therefore they may not be the major component linking SRIF receptor and voltage-gated K+ channels. It is likely that the observed increase in voltage-gated K+ currents in ovine somatotrophs is mediated by membrane-delimited signaling molecules, such as G proteins.

Several types of SRIF receptor have been cloned and classified as SSTR1-5, with SSTR2 being divided into SSTR2A and SSTR2B (22). SSTR2, SSTR4, and SSTR5 have been located in pituitary cells and are coupled to Gi-1, Gi-3, and Go proteins (22). These G proteins are presumably involved in various effects of SRIF, including a reduction in cAMP levels (12), an increase in K+ currents (6, 9), and a decrease in Ca2+ currents (4, 8). By use of relatively selective agonists for SSTR2 and SSTR5 receptors, these subtypes were shown to mediate the action of SRIF on Ca2+ channels (29). It is not clear so far which subtype or subtypes of SRIF receptor mediate the increase in K+ currents by SRIF. However, it is now clear that, in ovine somatotrophs, SRIF acts on voltage-gated Ca2+ and K+ channels via two different G proteins, Go-2 and Gi-3.

We conclude that the effect of SRIF on voltage-gated K+ currents in somatotrophs is mediated by Gi-3 proteins. Go, Gi-1, and Gi-2 proteins are not involved in the coupling of the increase in voltage-gated K+ currents by SRIF.

    ACKNOWLEDGEMENTS

I thank Dr. I. J. Clarke and Dr. P. D. Marley for critical reading and discussion of the manuscript, K. Loneragan for technical assistance, and S. Panckridge for preparing the graphics.

    FOOTNOTES

This work was funded by grants from the Australian National Health and Medical Research Council.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. Chen, Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, VIC 3168, Australia.

Received 25 February 1998; accepted in final form 21 April 1998.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(2):E278-E284
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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