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Somatostatin Increases Phospholipase D Activity and Phosphatidylinositol 4,5-bisphosphate Synthesis in Clonal Cells HIT-T15

Department of Biomedical Sciences, Iowa State University, Ames, Iowa

Received December 19, 2004; accepted March 22, 2005

	Abstract

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In the presence of arginine vasopressin (AVP), somatostatin increases [Ca²⁺]_i, leading to a transient increase in insulin release from clonal cells HIT-T15 via G_i/o and phospholipase C (PLC) pathway (Cheng et al., 2002a). The present study was to elucidate the mechanisms underlying somatostatin-induced [Ca²⁺]_i increase in the presence of AVP. We found that the effect of somatostatin was mediated by subunits but not by the subunit of G_i/o. Because somatostatin alone failed to increase [Ca²⁺]_i, we hypothesized that somatostatin increases phosphatidylinositol 4,5-bisphosphate (PIP₂) synthesis, providing extra substrate for preactivated PLC- to generate inositol 1,4,5-trisphosphate (IP₃). Somatostatin alone did not increase IP₃ levels, but AVP + somatostatin did. Somatostatin increased PIP₂ levels but decreased phosphatidylinositol 4-phosphate levels. We further hypothesized that PLD mediates somatostatin-induced changes in PIP₂ levels. Both the phospholipase D (PLD) inhibitors and antibody versus PLD1 antagonized AVP-somatostatin-induced increases in [Ca²⁺]_i. PLD inhibitor also antagonized somatostatin-induced increase in PIP₂ levels. In addition, somatostatin increased PLD activity. These results suggest that activation of somatostatin receptors that are coupled to the dimer of G_i/o led to PLD1 activation, thus promoting the synthesis of phosphatidic acid. Phosphatidic acid activates PIP-5 kinase, which evokes an increase in PIP₂ synthesis. The PIP₂ generated by somatostatin administration increases substrate for preactivated phospholipase C-, which hydrolyzes PIP₂ to form IP₃, leading to an increase in [Ca²⁺]_i. The regulation of PIP₂ synthesis by G_i/o-coupled receptors via PLD activation represents a novel signaling mechanism for somatostatin and a novel concept in the cross-talk between G_q- and G_i/o-coupled receptors in cells.

We reported recently that in the presence of AVP, somatostatin increased [Ca²⁺]_i and evoked a paradoxical, transient insulin release from HIT-T15 cells (Cheng et al., 2002a). Pretreatment with somatostatin for 100 s does not enhance AVP-induced increase in [Ca²⁺]_i (Cheng et al., 2002a). Others have found similar results using somatostatin as a G_i/o-coupled receptor agonist. For example, in SH-SY5Y cells, somatostatin increases [Ca²⁺]_i after pretreatment with carbachol, a cholinergic agonist, which signals via G_q (Connor et al., 1997). In another cell line, RINm5F, cyclopentyladenosine, a G_i/o-coupled receptor agonist, does not increase [Ca²⁺]_i when administered alone but increases [Ca²⁺]_i after pretreatment with carbachol (Biden and Browne, 1993). The cross-talk effects of somatostatin with AVP in HIT-T15 cells are mediated by G_i/o, the PLC pathway, and Ca²⁺ release from endoplasmic reticulum (Cheng et al., 2002a). The increase in [Ca²⁺]_i by somatostatin is attributable to a cross-talk between G_q and G_i/o (Cheng et al., 2002a). To date, most of the studies examining cross-talk signals between G_q- and G_i/o-coupled receptors have been performed through quantification of inositol phosphates and/or [Ca²⁺]_i (Selbie et al., 1997; Chan et al., 2000; Yeo et al., 2001). In addition, antibodies versus PLC- isozymes (Murthy et al., 1996) or PLC inhibitors such as U-73122 (Cheng et al., 2002a) have also been used to investigate the cross-talk mechanism of G_i/o-coupled somatostatin receptors. The results from some of these studies suggest that the dimer of G_i/o directly activates PLC- (Murthy et al., 1996; Chan et al., 2000). However, in our previous study, treatment with somatostatin alone, even at a high concentration of 1 µM, failed to increase [Ca²⁺]_i or insulin release (Cheng et al., 2002a). We therefore hypothesized that somatostatin increases PIP₂ synthesis in cells, thereby increasing the substrate for preactivated PLC- by AVP. In addition, we hypothesized that somatostatin activates PLD to increase the synthesis of phosphatidic acid, which in turn activates PIP-5 kinase to increase PIP₂ synthesis.

Our present findings indicate that somatostatin alone cannot increase IP₃ production. In addition, somatostatin-induced increase in IP₃ and [Ca²⁺]_i in the presence of AVP is mediated through the G-dimer of G_i/o, an increase in PLD activity, and a subsequent increase in PIP₂ synthesis.

	Materials and Methods

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Materials. All reagents were purchased from Sigma Chemical (St. Louis, MO), except that fura-2 acetoxymethyl ester (fura-2AM) was from Molecular Probes (Eugene, OR), and rabbit polyclonal antibodies versus G_i1/G_i2 and G_i3/G_o, and G were from BIOMOL Research Laboratories (Plymouth Meeting, PA), myo-[2-³H]inositol and [-³²P]ATP were from PerkinElmer Life and Analytical Sciences (Boston, MA).

Cell Culture and Transfection. HIT-T15 cells were maintained in RPMI 1640 medium with 10% FBS and aerated with 5% CO₂/95% air at 37°C. All experiments were performed using cells from passages 80 to 90. For overexpression of the C terminus of the -adrenergic receptor kinase (ct-ARK), HIT-T15 cells were transfected with pcDNAIIIB T8ark plasmid (donated by Dr. Silvio Gutkind, National Institutes of Health), using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.

Measurement of [Ca²⁺]_i in Single Cells. [Ca²⁺]_i was measured as described previously (ZhuGe and Hsu, 1995). Cells were loaded with 2 µM fura-2AM in Krebs-Ringer-bicarbonate buffer (KRB) for 30 min at 37°C. Measurement was performed in custom-made 35-mm culture dishes or a 20 x 20-mm perifusion chamber on the stage of an inverted fluorescence microscope (Carl Zeiss). Fluorescence images were obtained (_ex = 340 and 380 nm; _em = 510 ± 20 nm), and background was subtracted and divided on a pixel-by-pixel basis to generate spatially resolved maps of the [Ca²⁺]_i. The emitted signals were digitalized, recorded and processed using Attofluor Digital Fluorescence Imaging System (Atto Instruments, Rockville, MD). The [Ca²⁺]_i was calculated according to a method published previously (Grynkiewicz et al., 1985). Calibration was performed according the procedure provided by Attofluor, using fura-2 penta K⁺ as a standard.

Measurement of [Ca²⁺]_i in Transfected Cells. T8ARK-transfected HIT-T15 cells were seeded into black-walled, clear-based, poly-D-lysine–coated 96-well plates (Costar; Corning, Acton, MA) at a density of 10⁵ cells per well in RPMI 1640 medium, supplemented as described above, and cultured overnight. The cells were then incubated with the FLIPR Calcium 3 assay kit (Molecular Devices, Sunnyvale, CA) at 37°C for 60 min. After incubation, the plates were inserted into a fluorometric imaging plate reader (FLEXstationII; Molecular Devices, Sunnyvale, CA), and the fluorescence at _ex = 488 nm, and _em = 515 to 535 nm was used to monitor changes in [Ca²⁺]_i. Data were expressed as percentage increase of relative fluorescent units (RFU) divided by baseline RFU using Softmax Pro.

Microinjection Protocol. Single cells were grown for 2 days on glass coverslips of custom-made 35-mm culture dishes. Thereafter, cells were loaded with fura-2AM and mounted on the stage of an inverted microscope. For microinjection of antibodies, two cells from each dish were injected with intracellular buffer and rabbit antibodies, respectively, using a disposable glass pipette (VWR Scientific, West Chester, PA) held by a Narishige MW-3 micromanipulator. Pipettes were made by a PE-2 Micropipette puller (Narishige Scientific Instrument, Tokyo, Japan). All antibodies were diluted at 1:100 with intracellular buffer solution containing 27 mM K₂HPO₄, 8 mM NaH₂PO₄, and 26 mM KH₂PO₄, pH 7.3. Injection pressure was controlled by a pressure injection system (Picospritzer II; General Valve, Fairfield, NJ). A 30-min incubation period was allowed between the antibody injection and [Ca²⁺]_i measurement. For microinjection of PIP₂, one cell from each dish was injected with Na⁺ salt of PIP₂, which had been dissolved and further diluted in intracellular buffer at the respective PIP₂ concentration or buffer alone as the control. Microinjection of PIP₂, PIP, and phosphatidic acid (PA) was performed at 100 s after the perfusion with AVP into the culture dish. At the end of each experiment, the cell was depolarized with 10 mM KCl to test membrane integrity.

Determination of PIP and PIP₂. PIP and PIP₂ levels were measured using thin-layer chromatography as described previously (Norris and Majerus, 1994). HIT-T15 cells were labeled with 200 µCi/ml of [-³²P]ATP in phosphate-free KRB for 60 min and washed twice with centrifugation at 300g for 2 min. For experiments, cells were resuspended at a density of 15 x 10⁶ cells/ml/treatment. The reactions were terminated by addition of 1 ml of ice-cold 1 N HCl. Phospholipids were separated by a chloroform/methanol (1:1) mixture. The lower phase was dried under a stream of nitrogen, resuspended in 200 to 500 µl of chloroform/methanol (1:1) mixture, and spotted on silica gel plates. PIP and PIP₂ were identified by comigration with unlabeled standards, which were visualized by iodine staining and radiograph. One-centimeter blocks of the corresponding lanes for the samples were subsequently scraped, and radioactivity was quantified by liquid scintillation counting.

Determination of IP₃. Measurement of inositol phosphates followed modified procedures from a published report (Hoque et al., 2001). Cells were labeled with 20 µCi/ml of myo-[2-³H]inositol at 37°C for 90 min, by which time the incorporation of ³H into inositol lipids had reached a plateau (Hoque et al., 2001).

Cells were washed twice in phosphate-free KRB and centrifuged at 300g for 2 min. For experiments, cells were resuspended at a density of 20 x 10⁶ cells/ml/treatment. The reactions were terminated by addition of 0.5 ml of ice-cold 10% trichloroacetic acid, and samples were centrifuged at 3,000g for 20 min at 4°C. The supernatants were passed through a 200 to 400 mesh Dowex AG1-X 8 in formate form column (Bio-Rad Laboratories, Hercules, CA). Inositol phosphates were eluted by stepwise addition of 0.2, 0.5, and 1 M ammonium formate, which yielded inositol 1-monophosphate, inositol 1,4-bisphosphate, and IP₃, respectively. Radioactivity associated with IP₃ from each sample was quantified by liquid scintillation counting.

Determination of PLD Activity. PLD assay was performed using a method described previously (O'Launaigh et al., 2002). In brief, HIT-T15 cells were grown in 24-well plates overnight in RPMI 1640 medium with 10% FBS. The medium was then discarded, and 500 µl of 3 µCi/ml of [³H]myristic acid was added to each well for 60 min. Cells were then washed twice with KRB, and 400 µl of KRB was added to each well. Treatments were applied 15 s after the addition of 0.5% 1-butanol. The reactions were terminated 30 s after treatment had been administered. Phosphatidylcholine (PC) and phosphatidylbutanol were identified by migration with unlabeled standards, which were visualized by iodine staining and radiograph. One-centimeter blocks of the corresponding lanes for the samples were subsequently scraped and radioactivity was quantified by liquid scintillation counting.

Intracellular Delivery of Antibodies versus PLD. This technique was performed using BioPORTER protein delivery reagent (Gene Therapy Systems, San Diego, CA). Undiluted PLD antibodies (a gift from Dr. Sylvain Bourgoin of the University of Laval, Canada) were delivered into HIT-T15 cells following manufacturer's protocol. In brief, HIT-T15 cells were plated on a glass coverslip of custommade 35-mm culture dish. Antibody versus PLD (2 µg) (or normal rabbit plasma as control) was mixed with 1.5 µl of the protein delivery reagent and 150 µl of FBS-free RPMI medium and added to each well. The cells were incubated at 37°C for 4 h. The culture medium was then removed and replaced by KRB. Thereafter, cells were loaded with fura-2AM and determination of single cell [Ca²⁺]_i was performed as mentioned above.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Effect of antibodies versus Gi/o subunits on somatostatin (SS)-induced increase in [Ca2+]i in the presence of AVP in HIT-T15 cells. A, antibodies versus G, Gi1/Gi2, and Gi3/Go were diluted 1:100 and microinjected into single cells followed by a 30-min incubation period and Ca2+ image analysis. Somatostatin (100 nM) was given 100 s after administration of AVP (1 nM). Baseline [Ca2+]i were approximately 100 nM. Values are the mean ± S.E.; n = 10 cells/treatment from three independent cell preparations. *, p < 0.05 compared with somatostatin controls. B, representative calcium trace of microinjected antibodies versus G, in the presence of AVP, inhibited SS induced increase in [Ca2+]i. C and D, representative calcium traces of microinjected antibodies versus Gi1/Gi2 and Gi3/Go, respectively, in the presence of AVP, did not inhibit SS-induced increase in [Ca2+]i.

Data Analyses. All values are presented as mean ± S.E. Results were analyzed using analysis of variance and individual mean comparisons were made using the least significant difference test. The significance level was set at p < 0.05.

	Results

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Mediation by G of Somatostatin-Induced Increase in [Ca²⁺]_i in the Presence of AVP. The G_i/o- dimer has been demonstrated to be the essential subunit of the G protein that elicits this synergistic increase in [Ca²⁺]_i in the presence of G_q activation (Selbie et al., 1997; Quitterer and Lohse, 1999; Chan et al., 2000). To determine whether our cell system is also dependent on G_i/o- dimer, we microinjected antibodies versus G_i/o subunits into single cells in the presence of 1 nM AVP and then treated the cells with somatostatin (100 nM) to induce an increase in [Ca²⁺]_i. Antibody (1:100) versus G reduced the response to somatostatin by 89% (p < 0.05). In contrast, antibodies versus G_i1/G_i2 or G_i3/G_o (1:100) did not significantly change the response to somatostatin (Fig. 1). To further demonstrate the involvement of G_i/o- dimer, ct-ARK was expressed in HIT-T15 cells. Expressing ct-ARK has been shown to sequester G, thereby inhibiting its ability to stimulate downstream mediators (Inglese et al., 1992; Koch et al., 1994). Protein expression of the myc-tagged ct-ARK was determined by immunocytochemistry using anti-myc monoclonal antibody; 80 to 90% of HIT-T15 cells expressed the myc epitope (data not shown). When ct-ARK was expressed in HIT-T15 cells, it dramatically reduced somatostatin (100 nM)-induced increase in [Ca²⁺]_i in the presence of AVP (1 nM). In mock-transfected HIT-T15 cells, somatostatin in the presence of AVP caused a 81 ± 14% increase in RFU/baseline RFU, whereas cells that expressed ct-ARK elicited only a 14 ± 9% increase in RFU/baseline RFU when stimulated by AVP-somatostatin. All cells responded to 10 mM KCl by increasing [Ca²⁺]_i (data not shown). These results suggested that G_i/o- dimer is essential for the somatostatin-induced increase in [Ca²⁺]_i in the presence of AVP.

Somatostatin- and AVP-Induced Increase in IP₃ Levels. Somatostatin alone was unable to elicit any increase in [Ca²⁺]_i in HIT-T15 cells (Cheng et al., 2002a). We then hypothesized that somatostatin does not directly activate PLC-. We first determined the time course in which somatostatin + AVP produced the highest IP₃ levels. In the presence of AVP (1 nM), somatostatin (100 nM) increased IP₃ levels after 10 to 12 s of administration (Fig. 2A, p < 0.05). We then used this time frame to compare IP₃ levels among four treatment groups, terminating all reactions at 12 s after somatostatin administration. Somatostatin (100 nM) alone failed to increase IP₃ levels. AVP (1 nM) alone induced a small but significant increase in IP₃ levels (Fig. 2B). In cells pretreated with AVP (1 nM) for 100 s, followed by somatostatin (100 nM), IP₃ levels were the highest among four treatment groups. These results suggested that somatostatin alone cannot activate PLC- in HIT-T15 cells, because it did not increase IP₃ levels. AVP (1 nM) alone increased IP₃ levels in these cells, and concurrent administration of AVP and somatostatin caused a synergistic increase in IP₃ levels.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Somatostatin (SS)-induced increase in IP3 levels in the presence and absence of AVP in HIT-T15 cells. A, time course of AVP-somatostatin induced increase in IP3 levels, as determined by ion-exchange chromatography. Somatostatin (100 nM) was given 100 s after AVP (1 nM) and experiments terminated at the respective time; *, p < 0.05 compared with 0 s (n = 3). B, determination of IP3 levels among four treatment groups. Somatostatin (100 nM) was applied 100 s after AVP (1 nM); experiment was terminated at 12 s of somatostatin treatment; *, p < 0.05 compared with basal controls. Values are the mean ± S.E. (n = 4 independent cell preparations).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effect of PIP2 microinjection on [Ca2+]i in the presence of AVP in HIT-T15 cells. A, administration of PIP2 (1.5–50 amol) into single cells increased [Ca2+]i in a dose-dependent manner after 100 s of AVP (1 nM). Values are the mean ± S.E.; n = 3 independent cell preparations. B, representative calcium trace of HIT-T15 cells microinjected with intracellular buffer after the addition AVP did not increase [Ca2+]i. C, representative calcium trace of HIT-T15 cells microinjected with PIP2 alone at 50 amol did not increase [Ca2+]i. D, Representative calcium trace of HIT-T15 cells microinjected with 1.5 amol of PIP2 after the addition of AVP did not increase [Ca2+]i. E–G, representative calcium traces of microinjected PIP2 (5, 15, and 50 amol) did increase [Ca2+]i, while in the presence of AVP, in a dose-dependent manner.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Somatostatin (SS)-induced changes in PIP2 and PIP2 levels in HIT-T15 cells. PIP and PIP levels were determined by thin-layer chromatography. Values are the mean ± S.E. A, time course for changes in PIP and PIP2 levels by somatostatin in the presence of AVP. Somatostatin (100 nM) was administered 100 s after AVP (1 nM); the experiment was terminated at the indicated time; n = 3 independent cell preparations. *, p < 0.05 compared with 0 s. B and C, somatostatin-induced changes in PIP and PIP2 levels in the presence and absence of AVP. PIP2 (B) and PIP (C) levels were determined after exposure to different treatments. Somatostatin (100 nM) was applied 100 s after AVP (1 nM) and experiments terminated at 8 s after somatostatin administration; n = 4 independent cell preparations. *, p < 0.05 compared with basal controls.

Somatostatin-Induced Increase in PLD Activity. PIP₂ synthesis is catalyzed predominantly by PIP 5-kinase (Hawkins et al., 1992), which is activated by several input signals, including PLD (Jenkins et al., 1994). PLD converts PC into PA, which is known to activate PIP 5-kinase. We hypothesized that somatostatin stimulates PLD to increase PA formation, thereby activating PIP 5-kinase to increase PIP₂ synthesis. Somatostatin (100 nM) increased PLD activity by 2-fold over the control group (p < 0.05; Fig. 5A). In contrast, 100 nM AVP did not increase PLD activity (Fig. 5A). Pretreatment of HIT-T15 cells with pertussis toxin (PTX, 100 ng/ml) for 14 to 18 h abolished somatostatin (100 nM)-induced activation of PLD (p < 0.05; Fig. 5B), suggesting a G_i/o-coupled receptor-mediated response. HIT-T15 cells that expressed ct-ARK domain was able to abolish somatostatin (100 nM)-induced activation of PLD (p < 0.05, Fig. 5C). To demonstrate that activation of PLD can lead to an increase in PIP₂ levels, which can be used by PLC as a substrate to promote IP₃ production and a subsequent increase in [Ca²⁺]_i, we microinjected PA into HIT-T15 cells. Microinjection of PA (10 amol) in the absence of AVP (1 nM) produced no changes in [Ca²⁺]_I; however, in the presence of AVP, it increased [Ca²⁺]_i ([Ca²⁺]_i: PA, 2.1 ± 0.8 nM; AVP + PA, 39.1 ± 8.8 nM, n = 10 cells, p < 0.05). To determine whether somatostatin's ability to increase PLD activity has an impact on PIP₂ levels and its subsequent increase in [Ca²⁺]_i, we used 1-butanol, a PLD inhibitor, to determine its effects on somatostatin-induced increase in PIP₂ levels. Pretreatment of cells with 1-butanol (0.5%) abolished somatostatin-induced increase in PIP₂ levels (p < 0.05. Figure 6), but pretreatment with 2-butanol (0.5%), an inactive constitutional isomer of 1-butanol, did not (Fig. 6). To further demonstrate the involvement of PLD in AVP + somatostatin-induced increase in [Ca²⁺]_i, cells were pretreated with PLD inhibitor 1-butanol. 1-Butanol (0.5%) abolished AVP + somatostatin-induced increase in [Ca²⁺]_i, but 2-butanol (0.5%), did not (Fig. 7). Pretreatment with another PLD inhibitor, carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK, 10 µM) (Kessels et al., 1991) also abolished AVP + somatostatin-induced increase in [Ca²⁺]_i (Fig. 8). A high concentration of AVP (100 nM) caused an increase in both the control and zLYCK-pretreated cells (Fig. 8), suggesting that zLYCK does not inhibit G_q-PLC signaling pathway. To further classify PLD involvement in this pathway, we used antibodies versus PLD1 and PLD2, respectively, to determine which PLD isoform was responsible for the paradoxical increase in [Ca²⁺]_i caused by AVP-somatostatin. Only PLD1 was detected in HIT-T15 cells, using Western blot analysis (Fig. 9, inset). Antibody versus PLD1 inhibited somatostatin-induced [Ca²⁺]_i increase in the presence of AVP, whereas antibody versus PLD2 did not (Fig. 9). These results suggested that PLD1 mediates somatostatin-induced increases in PIP₂.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Somatostatin (SS)-induced increase in PLD activity in HIT-T15 cells. Experiments were terminated 30 s after somatostatin or AVP had been administered. A, effect of somatostatin and AVP. B, effect of PTX. HIT-T15 cells were pretreated overnight with PTX (100 ng/ml) before treatments. C, effect of ct-ARK expression on somatostatin-induced increase on PLD activity. HIT-T15 cells were transfected 24 h before PLD assay. Values are the mean ± S.E. (n = 4 independent cell preparations). *, p < 0.05 compared with controls. PBt, phosphatidylbutanol; PC, phosphatidylcholine.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Effect of 1-butanol and 2-butanol on somatostatin (SS)-induced increase in PIP2 levels. HIT-T15 cells were pretreated with 0.5% 1-butanol or 2-butanol 5 min before somatostatin treatment. Experiments were terminated at 8 s after somatostatin administration. Values are the mean ± S.E.; n = 4 independent cell preparations. *, p < 0.05 compared with basal controls.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effect of 1-butanol and 2-butanol on somatostatin (SS)-induced (100 nM) increase in [Ca2+]i in the presence of AVP (1 nM). HIT-T15 cells were pretreated with 1-butanol or 2-butanol (0.5%) 5 min before treatments. Cells treated with 1-butanol or 2-butanol were exposed to AVP (1 nM) for 150 s before the addition of somatostatin. Each line depicts the mean from 8 to 14 cells. The lines are representative of four independent experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Effect of zLYCK on somatostatin (SS)-induced (100 nM) increase in [Ca2+]i in the presence of AVP (1 nM). HIT-T15 cells were pretreated with zLYCK (10 µM) 60 min before treatments. Control and zLYCK-treated cells were exposed to AVP (1 nM) for 120 s before the addition of somatostatin. After the 60-s coexposure to AVP (1 nM) and somatostatin, cells were perfused with KRB for 120 s before the addition of 100 nM AVP. Each line depicts the mean from five to eight cells. The lines are representative of four independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 9. PLD immunoblot and effect of PLD antibodies on somatostatin (SS)-induced increase in [Ca2+]i in the presence of AVP. Inset, 10 µg of the whole cell protein was run on SDS-PAGE, transferred to the PVDF membrane, and blotted with PLD antibodies. For [Ca2+]i determination, HIT-T15 cells were pretreated with PLD antibodies using the BioPORTER protein delivery system 4 h before Fura-2AM loading. Cells were exposed to AVP (1 nM) for 120 s before the addition of somatostatin (100 nM). AVP (100 nM) was added 120 s after somatostatin treatment. Each line depicts the mean from five to eight cells. The lines are representative of four independent experiments.

	Discussion

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We reported previously that in clonal cells HIT-T15, somatostatin increased [Ca²⁺]_i and transiently stimulated insulin release in the presence of AVP (Cheng et al., 2002a). These effects of somatostatin in HIT-T15 cell are caused by activation of sstr2 (Cheng et al., 2002b) and are attributable to cross-talk between G_i/o and G_q (Cheng et al., 2002a). Cross-talk between G_q- and G_i/o-coupled receptors have been reported in other systems. For example, activation of G_i/o-coupled adenosine A₁-receptors enhances the stimulation of PLC- by G_q-coupled receptors such as ₁-adrenergic, bradykinin, histamine H₁, and muscarinic receptors (Selbie and Hill, 1998). For such cross-talk, activation of G_i/o alone usually has no effect, but it enhances G_q-mediated increases in PLC- activity, particularly when G_q is activated before G_i/o (Muller and Lohse, 1995; Connor et al., 1997; Quitterer and Lohse, 1999; Yeo et al., 2001). Activation of G_i/o-coupled adenosine A₁-receptors, ₂-adrenoceptors in COS cells (Quitterer and Lohse, 1999) or -or -opioid receptors in SH-SY5Y cells (Yeo et al., 2001) enhances inositol phosphate formation generated by activation of G_q-coupled receptors. In Chinese hamster ovary cells, neuropeptide Y, a G_i/o-coupled receptor agonist, enhances inositol phosphate formation generated by ATP, a G_q-coupled receptor agonist (Selbie et al., 1997). Somatostatin also increases [Ca²⁺]_i after activation of the G_q-coupled muscarinic receptors in SH-SY5Y cells (Connor et al., 1997).

So far, studies on the cross-talk between G_q and G_i/o indicate that G of G_i/o is responsible for the enhancement of G_q-generated signals (Selbie et al., 1997; Quitterer and Lohse, 1999; Chan et al., 2000). We demonstrated that antibody versus G nearly abolished somatostatin-induced increase in [Ca²⁺]_i, whereas antibodies versus G_i1/G_i2 and G_i3/G_o failed to do so. In addition, overexpressing ct-ARK, which binds avidly to G (Inglese et al., 1992; Koch et al., 1994), greatly reduced somatostatin-induced increases in [Ca²⁺]_i. Our findings are consistent with what is found in the literature (Selbie et al., 1997; Quitterer and Lohse, 1999; Chan et al., 2000) and further suggest that in cells, the increase in [Ca²⁺]_i by somatostatin is mediated through G.

Several studies have shown that G of G_i/o can activate a number of enzymes, including PLC- (Blake et al., 2001), PLA₂ (Kim et al., 1989), mitogen-activated protein kinase (Koch et al., 1994), Raf-1 (Pumiglia et al., 1995), ARK (Goldman et al., 1997), phosphatidylinositol 3-kinase (Lopez-Ilasaca et al., 1998), and adenylyl cyclase (Myung and Garrison, 2000). In intestinal smooth muscle cells, somatostatin alone activates PLC-₃, thereby increasing IP₃ levels, [Ca²⁺]_I, and contractions through the G of G_i/o (Murthy et al., 1996).

Although others have suggested that enhancement of G_q signals by G_i/o occurs through activation of PLC- (Chan et al., 2000) or interaction with a step after PLC activation (Yeo et al., 2001), none of them has attributed the effect of G_i/o to a step before PLC activation, except that Schmidt et al. (1996 and 1998) suggested that G_i/o mediates an increase in PIP₂ levels. In the present study with HIT-T15 cells, after somatostatin treatment, PIP levels decreased but PIP₂ levels increased, suggesting that somatostatin may increase PIP₂ synthesis. Thus, we have suggested for the first time that somatostatin can increase PIP₂ synthesis, which in turn provides extra substrate for preactivated PLC- by AVP to generate high levels of IP₃. Without a preactivated PLC-, somatostatin failed to increase IP₃ levels (Fig. 3) and [Ca²⁺]_i (Cheng et al., 2002a). However, we cannot rule out the possibility that the increase in PIP₂ and decrease in PIP levels might have been caused by a decrease in phosphatase activity. In addition, because the IP₃ assay that we used in the present study could not differentiate inositol 1,3,4-trisphosphate from 1,4,5-trisphosphate, some of the IP₃ observed in the present study could be attributable to inositol 1,3,4-trisphosphate. Nevertheless, our results are consistent with the findings using cyclopentyladenosine, an adenosine A₁-receptor agonist, in RINm5F cells, another cell line (Biden and Browne, 1993). In RINm5F cells, activation of adenosine A₁-receptors alone fails to increase IP₃ levels and [Ca²⁺]_i but increases them after activation of M₃ receptors (Biden and Browne, 1993). Our findings are further supported by those of the administration of PIP₂ into the cells, in which PIP₂ alone failed to increase [Ca²⁺]_i, but PIP₂ in the presence of a small concentration of AVP (1 nM) increased [Ca²⁺]_i. Because PIP₂ and PIP failed to increase [Ca²⁺]_i in the absence of AVP (1 nM), we believe that these phospholipids are unable to elicit any [Ca²⁺]_i response in HIT-T15 cells. The failure of microinjected PIP (50 amol) to induce changes in [Ca²⁺]_i in the presence of AVP (1 nM) demonstrated that the microinjection of PIP₂ in the presence of AVP, which caused an increase in [Ca²⁺]_i, was caused by the use of PIP₂ as a substrate for low-grade PLC activation and was not an artifact of the system. In HEK 293 cells with stable expression of M₃ receptors, carbachol increases PIP₂ levels for at least 30 min (Schmidt et al., 1996). The M₃ receptor-mediated increase in PIP₂ levels is attributable to G_i-protein coupling, because this effect was inhibited by PTX pretreatment. In HEK 293 cells, activation of plasma membrane purinergic receptors and lysophosphatidic acid receptors, respectively, also increases PIP₂ levels for 40 min (Schmidt et al., 1998). These findings are somewhat different from those of ours, because somatostatin-induced increase in PIP₂ levels in HIT-T15 cells lasted < 12 s, but the effect of G_i/o-coupled receptor agonists in HEK 293 cells lasted 40 min. It is not clear why these two systems are so much different in terms of the duration of the agonist-induced PIP₂ levels. However, in the study with HEK 293 cells, for preactivation of PLC, Schmidt et al. (1998) used G_q-coupled receptor agonists, which markedly lowered PIP₂ levels. Upon removal of the agonists, the PIP₂ levels increased again, which were above the initial control levels (K. H. Jakobs, personal communication).

View larger version (15K): [in this window] [in a new window]   Fig. 10. Summary of the cross-talk between AVP receptor (V1b) and somatostatin receptor (sstr2) in stimulation of insulin release from cells. Activation of the Gi/o-coupled receptor by somatostatin increases PIP2 synthesis from PIP through G. G activates PLD to increase the synthesis of phosphatidic acid (PA) from phosphatidylcholine (PC). PA activates PIP 5-kinase to increase synthesis of PIP2, providing extra substrate for preactivated PLC- by AVP. These increases in DAG and IP3 levels and [Ca2+]i lead to insulin release. ER, endoplasmic reticulum.

In this system, somatostatin was able to increase PIP₂ levels, providing extra substrate for PLC-. On the other hand, PIP₂ can regulate a wide range of cellular processes, including exocytosis (Cremona and De Camilli, 2001), clathrin-mediated endocytosis (Gillooly and Stenmark, 2001), actin rearrangement (Tolias et al., 2000), vesicle docking (Brown et al., 2001), opening of G-protein-gated inwardly rectifying K⁺ channels (Zhang et al., 1999), K_ATP channels (Baukrowitz et al., 1998), and membrane ruffling and trafficking (Honda et al., 1999). Because of the diverse role of PIP₂ in cellular processes, the effect of somatostatin-induced increase in PLD activity and its subsequent increase in PIP₂ levels on normal physiology of cells is uncertain.

In the present study, when AVP was needed to cause a low-grade PLC activation, we used 1 nM AVP in most of the experiments. This concentration of AVP usually caused an increase in [Ca²⁺]_i of 0 to 50 nM, which was consistent with our previous study (Cheng et al., 2002a). However, in the experiment in which butanols were studied (Fig. 7), AVP at 1 nM increased [Ca²⁺]_i by 90 nM. In this particular experiment, probably as a result of different FBS used in cell culture, the cells became more responsive to AVP than usual. As a result of the hyper-response to AVP, somatostatin-induced [Ca²⁺]_i increase was attenuated. This phenomenon was also seen in our previous study (Cheng et al., 2002a).

In summary, we have demonstrated a novel signaling mechanism for somatostatin. The activation of somatostatin receptors, which are coupled to G_i/o, leads to an increase in PIP₂ synthesis through G activation of PLD. The PIP₂ generated by somatostatin administration provides extra substrate for preactivated PLC-, which hydrolyzes PIP₂, thereby increasing IP₃ levels, [Ca²⁺]_i, and a transient release of insulin from HIT-T15 cells (Fig. 10). This is the first report regarding somatostatin-induced increase in PLD activity and PIP₂ synthesis by activation of G_i/o-coupled receptors.

	Acknowledgements

 
We greatly appreciate the generous donation of pcDNAIIIB T8ARK plasmid by Dr. Silvio Gutkind of National Institute of Dental and Craniofacial Research, National Institutes of Health, and antibodies versus PLD1 and PLD 2 by Dr. Sylvain Bourgoin of the University of Laval, Canada. We would also like to acknowledge Dr. Guangxing Bai for contribution to Western blot analysis.

	Footnotes

 
H.C. and J.A.G. contributed equally to the present work.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.104.010470.

ABBREVIATIONS: GPCR, G protein-coupled receptor; PLC, phospholipase C; PLD, phospholipase D; PIP₂, phosphatidylinositol 4,5-bisphosphate; IP₃, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; ER, endoplasmic reticulum; AVP, arginine vasopressin; U-73122, 1-[6-[[17-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; AM, acetoxymethyl ester; ct-ARK, C terminus of the -adrenergic receptor kinase; KRB, Krebs-Ringer-bicarbonate buffer; RFU, relative fluorescent unit; PIP, phosphatidylinositol 4-phosphate; PA, phosphatidic acid; PC, phosphatidylcholine; PTX, pertussis toxin; zLYCK, carbobenzyloxy-leucine-tyrosine-chloromethylketone; HEK, human embryonic kidney; PVDF, polyvinylidene difluoride.

Address correspondence to: Dr. Walter H. Hsu, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011-1250. E-mail: whsu{at}iastate.edu

	References

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