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Endocrinology Vol. 143, No. 3 889-897
Copyright © 2002 by The Endocrine Society

GRH-SOMATOSTATIN-GH

Somatostatin Stimulates GH Secretion in Two Porcine Somatotrope Subpopulations through a cAMP-Dependent Pathway

José L. Ramírez1, Francisco Gracia-Navarro, Socorro García-Navarro, Rafael Torronteras2, María M. Malagón and Justo P. Castaño

Department of Cell Biology, Physiology, and Immunology, University of Cordoba, E-14071 Cordoba, Spain

Address all correspondence and requests for reprints to: Dr. Francisco Gracia-Navarro, Department of Cell Biology, Physiology, and Immunology, Campus de Rabanales, Edificio C-6, Planta 3, University of Cordoba, E-14071 Cordoba, Spain. E-mail: . fgracia{at}uco.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Somatostatin (SRIF) inhibits GH release from rat somatotropes by reducing adenylate cyclase (AC) activity and the free cytosolic calcium concentration ([Ca2+]i). In contrast, we have reported that SRIF can stimulate GH release in vitro from pig somatotropes. Specifically, 10-7 and 10-15 M SRIF stimulate GH release from a subpopulation of high density (HD) somatotropes isolated by Percoll gradient centrifugation, whereas in low density (LD) somatotropes only 10-15 M SRIF induces such an effect. To ascertain the signaling pathways underlying this phenomenon, we assessed SRIF effects on second messengers in cultured LD and HD cells by measuring cAMP, IP turnover, and [Ca2+]i. Likewise, contribution of the corresponding signaling pathways to SRIF-induced GH release was evaluated by blocking AC, PLC, extracellular Ca2+ influx, or intracellular Ca2+ mobilization. Both 10-7 and 10-15 M SRIF increased cAMP, IP turnover, and [Ca2+]i in HD cells. Conversely, in LD cells 10-7 M SRIF reduced [Ca2+]i, but did not alter cAMP or IP, and 10-15 M SRIF was without effect. Interestingly, SRIF-stimulated GH release was abolished in both subpopulations by AC blockade, but not by PLC inhibition. Furthermore, SRIF-induced GH release was not reduced by blockade of extracellular Ca2+ influx through voltage-sensitive channels or by depletion of thapsigargin-sensitive intracellular Ca2+ stores. Therefore, SRIF stimulates GH secretion from cultured porcine somatotrope subpopulations through an AC/cAMP pathway-dependent mechanism that is seemingly independent of net increases in IP turnover or [Ca2+]i. These novel actions challenge classic views of SRIF as a mere inhibitor for somatotropes and suggest that it may exert a more complex, dual function in the control of porcine GH release, wherein molecular heterogeneity of somatotropes would play a critical role.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT HAS BEEN classically accepted that the primary regulation of GH secretion from pituitary somatotropes is the result of an interplay between two hypothalamic factors, the stimulatory GHRH and the inhibitory somatostatin (SRIF) (1, 2, 3). However, results obtained in several species, including sheep (4), pigs (5), and humans (6), have suggested that the mode of action of the tandem GHRH/SRIF might be more complex than initially envisioned in the well known male rat model. In fact, recent studies have provided compelling evidence that additional factors, such as the newly identified peptide ghrelin (7), are required to fully elucidate the primary hypothalamic regulation of GH secretion.

The discovery of additional mechanisms of action for known peptides, such as SRIF, may also contribute to a better understanding of this regulation. In this regard and despite the common view that SRIF only functions as a GH inhibitor, previous results from our group and others have suggested that this peptide may act not only as an inhibitor but also as a stimulator of GH release in the pig. Thus, whereas SRIF administration did not decrease basal GH in vivo in fetal (8) or prepubertal (9) gilts, continuous SRIF infusion enhanced the stimulatory effect of GHRH in this latter model (9). In vitro, SRIF has been found to evoke paradoxical stimulatory GH responses in superfused anterior pituitary fragments from peripubertal Göttingen miniature sows (10) as well as from individual fetal and neonatal (but not peripubertal) German Landrace pigs (10, 11). In line with these findings, we observed that SRIF stimulates GH secretion from cultured porcine pituitary cells when administered at low doses (12). A possible clue to the cellular mechanisms underlying these responses was gained in the process of studying the heterogeneous nature of the porcine somatotrope population. To be more specific, we found that two subpopulations of low (LD) and high (HD) density somatotropes separated by Percoll gradient centrifugation displayed distinct ultrastructure and differed in their secretory response to GHRH and SRIF. In particular, we observed that 10-7 M SRIF was able to reduce GHRH-induced, but not basal, GH release in LD somatotropes, whereas this same SRIF concentration did not alter the effect of GHRH from HD somatotropes and, conversely, stimulated GH secretion from this cell subset when administered alone (13). Subsequent studies revealed that this is a specific effect induced by SRIF directly on somatotropes, and that it is dose dependent; SRIF stimulated GH release at both high (10-7 M) and low (10-15 M) doses in HD somatotropes, whereas in LD somatotropes it only induced this effect at low doses (14). The unique features of these effects led us to question what could be the subcellular mechanisms that mediate the stimulatory response of porcine somatotropes to SRIF. To answer this question, we decided to investigate the role of distinct second messenger pathways in the responses of LD and HD porcine somatotropes to SRIF.

A number of studies have established that SRIF acts through binding to specific membrane receptors that belong to the superfamily of G protein-linked receptors with seven transmembrane-spanning regions (15, 16, 17). Five receptor subtypes, named SSTR1–5, have been cloned to date in human and rodents, and all five have been shown to be present, albeit in distinct proportions, in rat somatotropes (18). Activation of SSTRs by SRIF induces different cellular processes that vary depending on the cell type and the SSTR subtype considered (15, 16, 17). Nevertheless, all SSTRs have been shown to be able to associate with inhibitory pertussis toxin-sensitive G proteins (Gi), and to inhibit adenylate cyclase (AC) activity, one of the hallmarks of SRIF action in somatotropes and other cell types (15, 16, 17, 19). Indeed, it has been described that SRIF decreases both basal and stimulated cAMP levels in rat and human pituitary cell cultures (20, 21, 22, 23). Likewise, SRIF inhibits basal and GHRH-induced free cytosolic calcium concentration ([Ca2+]i) through a mechanism that involves Gi/Go proteins, K+ channel-activated hyperpolarization, and inhibition of extracellular Ca2+ influx through voltage-sensitive Ca2+ channels (VSCC) (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). In contrast, the role of the PLC/IP/PKC pathway in mediating the SRIF action in somatotropes is less clear, although it has been reported that SRIF inhibits GH release induced by phorbol esters (31, 32) and by synthetic GH secretagogues known to act through the PLC/IP/PKC pathway (33, 34). Accordingly, in the present study we have analyzed the possible involvement of cAMP-, IP-, and Ca2+-dependent mechanisms in the secretory response of LD and HD porcine somatotrope subpopulations to SRIF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, cell dispersion, separation of subpopulations, and cell culture
Prepubertal female Large-White/Landrace pigs (5–6 months old) were obtained from a local slaughterhouse. Animals were killed by exanguination after brief electrical stunning, and pituitary glands were immediately removed and transferred to sterile cold (4 C) medium (D-valine modified MEM, Sigma, London, UK) supplemented with 0.1% BSA (Sigma) and antibiotic-antimycotic solution (Sigma). In the laboratory, pituitaries were washed twice with fresh medium, and the posterior lobes were discarded. For each separate, independent experiment, six to eight glands were pooled and dispersed together by means of an enzymatic-mechanical dispersion method described in detail previously (13, 14, 35). Thereafter, 30–40 x 106 dispersed cells were centrifuged (3000 x g, 25 min) in a hyperbolic, continuous Percoll density gradient (Pharmacia LKB, Uppsala, Sweden) to separate the two subpopulations of porcine somatotropes of low (LD; 1.051–1.064 g/ml) and high (HD; 1.087–1.098 g/ml) density, which have been thoroughly characterized in previous studies (13, 14, 35, 36, 37, 38). Monodispersed cells from each subpopulation were cultured (37 C, 5% CO2) in MEM supplemented with 10% FBS (Sera-Lab, Crawley-Down, UK). Experiments were carried out after 4 d of culture. A 2-h preincubation in fresh MEM without FBS was used to stabilize basal GH secretion before adding test substances.

A two-pronged approach was used to ascertain the possible involvement of distinct second messenger pathways in the responses of LD and HD porcine pituitary cells to two distinct, representative doses of SRIF [high (10-7 M) and low (10-15 M)]. These concentrations were selected based on a previous dose-response study (14), because they ensure that both opposite (using 10-7 M SRIF) and similar (using 10-15 M SRIF) secretory responses of LD and HD somatotropes to this peptide could be analyzed and compared. In the first approach, we assessed the direct effects of both SRIF doses on cAMP levels, IP turnover, and [Ca2+]i in separated LD and HD cells. Secondly, we evaluated the relative contributions of the corresponding intracellular signaling pathways to the secretory response of somatotropes by measuring the effects of both SRIF doses on GH release from LD and HD cell cultures after selectively inhibiting the key enzymes AC and PLC and after blocking extracellular Ca2+ influx or depleting intracellular Ca2+ stores.

cAMP measurement
To measure intracellular cAMP accumulation, dispersed cells from each subpopulation were plated in six-well tissue culture plates at a density of 2 x 106 cell/well in 2 ml MEM-FBS. After the 2-h preincubation in FBS-free MEM, cells were incubated for 30 min in MEM containing 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; Sigma) to prevent enzymatic degradation of cAMP. Then, cells were incubated for an additional 30-min period in MEM-IBMX in the presence or absence of SRIF (UCB Bioproducts, Brain L’Alleud, Belgium) at high (10-7 M) or low (10-15 M) concentration. Medium was then aspirated, and wells were scraped in 0.01 M PBS with 4 mM EDTA. Aliquots for determination of protein were removed, and samples were sonicated (3 min), boiled (5 min), and centrifuged (10,000 rpm, 5 min). The supernatant was removed and stored at -20 C until cAMP determination by means of a [3H]cAMP assay kit (Amersham Pharmacia Biotech, Aylesbury, UK). Results are reported as picomoles of cAMP per mg total protein.

[3H]Myo-inositol incorporation measurement
To determine [3H]myo-inositol incorporation, LD and HD cells were plated in 12-well tissue culture plates at a density of 6 x 105 cell/well·2 ml MEM-FBS and cultured during 4 d. After the 2-h preincubation in FBS-free medium, cells were incubated in the presence or absence of 10-7 or 10-15 M SRIF for 30 min in 500 µl MEM containing [3H]myo-inositol (3 µCi/ml; 10–25 Ci/mmol; DuPont, Stevenage, UK). Then, medium was aspirated, and the reaction was stopped by adding 500 µl ice-cold 20% trichloroacetic acid. Wells were scraped, and the contents were placed into Eppendorf tubes, sonicated, and pelleted by centrifugation at 12,000 rpm for 15 min at 4 C. Supernatant, which contained IPs, was removed and measured in a scintillation counter (LS 6000TA, Beckman Coulter, Inc., Fullerton, CA). Chloroform/methanol (1:1, vol/vol) was added to the pellet to extract the phosphoinositides (PIPs). After centrifugation (12,000 rpm, 20 min), the supernatant was also removed and measured. Because the results obtained for IPs and PIPs showed identical profiles and statistical differences (despite the expected differences in the absolute counts per min measurements), both values were added, and the results are reported as total amount of [3H]myo-inositol incorporated in each sample (IPs plus PIPs) and expressed as counts per min/mg total protein.

[Ca2+]i measurement
Monodispersed cells from LD and HD subpopulations were separately plated on microgrid coverslips (Eppendorf, Netheler, Germany) at a density of 25,000 cells/coverslip, placed onto 24-well culture plates, and incubated in MEM-FBS for 3–5 d before microfluorometric evaluation of [Ca2+]i using the fluorescent probe indo-1 as previously described in detail (37). Briefly, cells were loaded (30 min; 37 C) in MEM containing 5 µM indo-1 penta-acetoxymethyl ester and 0.02% (vol/vol) Pluronic F127 (Molecular Probes, Inc., Eugene, OR), washed twice with fresh medium, and incubated for an additional 30-min period to allow for the complete Indo-1 hydrolysis. [Ca2+]i was monitored by a dual emission microfluorometric system (Nikon Corp., Tokyo, Japan). The fluorescence emission of indo-1 induced by excitation at 355 nm was recorded at two wavelengths (405 nm, calcium-indo-1 complex form; 485 nm, free indo-1 form) by separate photometers (Nikon). Both 405 and 485 nm signals, and the 405/485 ratio were continuously monitored and stored using the software FASTINCA 1.03 (Nikon). Absolute [Ca2+]i levels were calculated using the Grynkiewicz formula (39) as described previously (37). The effect of SRIF on [Ca2+]i was tested by applying the peptide in the vicinity of single, randomly selected cells for 5 sec by means of a pressure ejection system (Nikon). To counteract the rapid dissolution of the peptide in the medium during the pulse, the concentrations of SRIF tested were 100-fold higher (10-5 or 10-13 M) than those employed in the cAMP, IP, and GH release experiments. The precise localization of each measured cell on the alphanumeric grid of the photoetched coverslip was recorded, and somatotropes were identified after porcine GH (pGH) immunostaining as previously described (37).

Evaluation of pGH release
In this set of experiments cells from each subpopulation were cultured for 4 d at a density of 3 x 105 cells/well·ml MEM-FBS in 24-well plates. After the 2-h preincubation in FBS-free medium, cells were pretreated for an additional 2-h period with MEM alone or MEM containing one of the following inhibitors: 1) AC inhibitor, 10 µM MDL-12,330A (Research Biochemical International, Natick, MA) (38); 2) PLC inhibitor, 50 µM U-73122 (Research Biochemical International) (38); 3) VSCC blocker, 2 mM CoCl2 (26, 38); and 4) sarco/endoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor (to deplete intracellular Ca2+ stores), 100 nM thapsigargin (Sigma) (38). Thereafter, cells were challenged with 10-7 or 10-15 M SRIF during 30 min in the presence or absence of the corresponding inhibitor. Finally, medium was aspirated from each well and microfuged for 5 min, and the supernatant was stored at -20 C until released porcine GH was measured by a homologous enzyme immunoassay (EIA) procedure as previously described (14). Pig GH (USDA-B-1, AFP-11716C; supplied by Dr. A. F. Parlow, Pituitary Hormones and Antisera Center, Harbor University of California-Los Angeles Medical Center, Los Angeles, CA) was used in the EIA for both plate coating and standard curve, and a specific anti-pGH developed in rabbit (University of California, Berkeley, CA) was used as primary antiserum, at a dilution of 1:200,000. The sensitivity of the EIA was 0.65 ± 0.12 ng pGH/ml.

Statistical analysis
Data are expressed as the mean ± SEM obtained from at least three separate, independent experiments. A minimum of four replicate wells per treatment were tested in each experiment. Statistical analysis was carried out using a one-way ANOVA, followed by a statistical test for multiple comparisons (Duncan’s multiple range test and critical ranges) by use of the software package Statistica (StatSoft, Inc., Tulsa, OK). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of the cAMP-dependent pathway in SRIF-stimulated GH release
Measurement of intracellular cAMP accumulation in separate porcine pituitary cell subpopulations confirmed our previous observation that, under basal culture conditions, cAMP levels are higher in LD than in HD cells (Fig. 1Go) (38). Interestingly, a 30-min treatment with SRIF evoked a marked increase in cAMP content in HD cells, which was of comparable magnitude (3- to 4-fold over control) at the two doses of the peptide tested (Fig. 1Go, lower panel). On the other hand, 10-7 M SRIF did not modify intracellular cAMP content in LD cell cultures, and 10-15 M of this peptide only induced a slight increase in cAMP that did not reach statistical significance (Fig. 1Go, upper panel). To determine the possible contribution of such cAMP rises to the SRIF-induced GH release, we evaluated the effect of the specific AC inhibitor MDL-12,330A on the secretory response of somatotropes. Neither this nor the rest of the inhibitors used in the present study to analyze intracellular signaling pathways significantly altered basal GH secretion from cultured somatotrope subpopulations. In contrast, as shown in Fig. 2Go, treatment with MDL-12,330A completely abolished the stimulation of GH release evoked by 10-15 M SRIF in LD cells (Fig. 2Go, upper panel), as well as the stimulatory response induced by both doses of SRIF in HD somatotropes (Fig. 2Go, lower panel). Thus, these results indicate that the activation of the AC/cAMP system contributes essentially to the SRIF-stimulated GH secretion from porcine somatotrope subpopulations.



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  		Figure 1. Effects of 10-7 or 10-15 M SRIF on intracellular cAMP accumulation in monolayer cultures of porcine LD (upper panel) and HD (lower panel) cells. After 4 d of culture in MEM-FBS, cells were equilibrated for 2 h in serum-free medium and then incubated with 1 mM IBMX during 30 min. Then cells were challenged with SRIF in the presence of IBMX and incubated for 30 min. Thereafter, cAMP production was measured. Data are expressed as picomoles of cAMP per mg total protein. Each bar represents the mean ± SEM of four independent experiments. At least three replicate wells were evaluated per treatment in each experiment. a, P < 0.05 vs. corresponding control.

 


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  		Figure 2. Effect of AC inhibition on SRIF-induced GH secretion in LD (upper panel) and HD (lower panel) porcine somatotropes. After 4 d of culture in MEM-FBS, cells were equilibrated for 2 h in serum-free MEM and then incubated with or without 10 µM MDL-12,330A. Thereafter, cells were challenged with 10-7 or 10-15 M SRIF during 30 min in the presence or absence of MDL-12,330A. Each bar represents the mean ± SEM of five independent experiments. At least four replicate wells were evaluated per treatment in each experiment. Data are expressed as a percentage of the respective control value (100%; dotted line). a, P < 0.05 vs. corresponding control; b, P < 0.05 vs. corresponding SRIF dose alone.

 
Role of the IP-dependent pathway in SRIF-stimulated GH release
As for cAMP, levels of [3H]myo-inositol incorporation under basal culture conditions were expectedly higher for LD than for HD cells (Fig. 3Go). None of the SRIF doses tested significantly altered [3H]myo-inositol uptake in LD cell cultures (Fig. 3Go, upper panel). Conversely, both 10-7 and 10-15 M SRIF similarly increased [3H]myo-inositol uptake in cultures of HD cells (Fig. 3Go, lower panel). The PLC inhibitor U-73122 was applied to ascertain the possible contribution of the PLC-IP pathway to the stimulation of the GH secretory response caused by SRIF in porcine somatotrope subpopulations. Interestingly, despite the increase in [3H]myo-inositol incorporation caused by SRIF in HD cells, the presence of the PLC inhibitor did not affect SRIF-stimulated GH secretion from HD somatotropes, regardless of the SRIF dose considered (Fig. 4Go). Similarly, U-73122 did not modify the increased GH release caused by 10-15 M SRIF in LD somatotropes. According to these results, activation of the PLC-IP intracellular signaling pathway by SRIF does not appear to be required for the stimulatory action of this peptide on GH release from porcine somatotrope subpopulations.



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  		Figure 3. Incorporation of [3H]myo-inositol in cultures of porcine LD (upper panel) and HD (lower panel) cells in response to 10-7 or 10-15 M SRIF. After 4 d of culture in MEM-FBS, cells were equilibrated for 2 h in serum-free medium and then incubated with SRIF for 30 min in the presence of [3H]myo-inositol. At the end of the incubation, samples were recovered, and labeled IPs and PIPs were measured as described in Materials and Methods. Data are expressed as the total counts per min/mg total protein. Each bar represents the mean ± SEM of three independent experiments. At least three replicate wells were evaluated per treatment in each experiment. a, P < 0.05 vs. corresponding control.

 


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  		Figure 4. Secretory response of LD (upper panel) and HD (lower panel) porcine somatotropes to SRIF after blockade of PLC activity. After 4 d of culture in MEM-FBS, cells were equilibrated for 2 h in serum-free MEM and then incubated with or without 50 µM U-73122. Thereafter, cells were incubated with 10-7 or 10-15 M SRIF for 30 min in the presence or absence of U-73122 (n = 6). See Fig. 2Go for further details. a, P < 0.05 vs. corresponding control.

 
Role of [Ca2+]i in SRIF-stimulated GH release
Microfluorimetric evaluation of [Ca2+]i in response to a 5-sec SRIF pulse was carried out in 85 LD and 66 HD single somatotropes obtained from 6 independent experiments, and the main features of such responses are summarized in Table 1Go. As expected, basal [Ca2+]i (40–60 sec recording prior SRIF pulse) was higher in LD than in HD somatotropes (37). The proportion of LD somatotropes showing a profile of response was similar after treatment with 10-5 or 10-13 M SRIF, yet only the higher dose caused a significant reduction in basal [Ca2+]i in this somatotrope subset (Table 1Go and Fig. 5Go, A and B). In marked contrast, both SRIF doses evoked a similar and modest (albeit significant) [Ca2+]i increase in HD somatotropes, which showed a higher responsiveness to low-dose SRIF (Table 1Go and Fig. 5Go, C and D).


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  		Table 1. Parameters of the [Ca2+]i response of LD and HD somatotropes to 10-5 and 10-13 M SRIF

 


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  		Figure 5. Representative profiles of the changes induced by 10-5 (A and C) and 10-13 M (B and D) SRIF in [Ca2+]i in LD somatotropes (A and B) and HD somatotropes (C and D). Arrows indicate the moment of the 5-sec SRIF pulse administered with a pressure ejection system. To counteract the rapid dissolution of the peptide in the medium during the pulse, the concentrations of SRIF tested were 100-fold higher (10-5 or 10-13 M) than those employed in the cAMP, IP, and GH release experiments. In LD cells, 10-5 M SRIF caused a significant decrease in [Ca2+]i (A), whereas 10-13 M SRIF only evoked slight reductions that were not significant (B). On the other hand, 10-5 M SRIF increased [Ca2+]i in 36% of HD somatotropes (C), and 10-13 M SRIF evoked similar increases in [Ca2+]i (D) in 57% of HD somatotropes.

 
We next investigated the possible relationship of these SRIF-induced changes in [Ca2+]i with the GH secretory response of LD and HD somatotropes to SRIF, as well as the relative contribution of extra- and intracellular Ca2+ sources to these responses. As illustrated in Fig. 6Go, blockade of extracellular Ca2+ entry through VSCC with CoCl2 did not modify any of the stimulatory responses induced by SRIF in LD and HD somatotropes. Likewise, depletion of thapsigargin-dependent intracellular Ca2+ stores did not affect the increase in GH release evoked by SRIF in both LD (Fig. 7Go, upper panel) and HD (Fig. 7Go, lower panel) somatotropes. Thus, when the moderate SRIF-evoked [Ca2+]i changes and the lack of effect of extra- and intracellular Ca2+ inhibition in SRIF-induced GH release are viewed together, our results comprise compelling evidence that net increases in [Ca2+]i are not required for SRIF to stimulate GH secretion from porcine LD and HD somatotropes.



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  		Figure 6. Effect of blockade of extracellular Ca2+ influx through VSCC by 2 mM CoCl2 on SRIF-stimulated GH secretion in LD (upper panel) and HD (lower panel) porcine somatotropes (n = 3). See Fig. 2Go for further details. a, P < 0.05 vs. corresponding control.

 


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  		Figure 7. Effect of depletion of intracellular Ca2+ stores by thapsigargin (100 nM) on the SRIF-stimulated GH secretion in LD (upper panel) and HD (lower panel) porcine somatotropes (n = 3). See Fig. 2Go for further details. a, P < 0.05 vs. corresponding control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that SRIF constitutes one of the major and best known inhibitory inputs in the entire neuroendocrine system (15, 16, 17, 40). In contrast, its stimulatory actions are relatively scarce and less understood. In somatotropes, available information supports the common view that SRIF solely acts as the major inhibitory signal for GH secretion (1, 2). However, we recently reported that SRIF can act as a dual stimulatory/inhibitory factor in cultured pig somatotropes, and that such dual action is related to the dose of SRIF and to the presence within somatotropes of two cell subpopulations (LD and HD) with different response to SRIF (12, 13, 14). In an attempt to elucidate the signaling mechanisms underlying this unusual GH-releasing ability of SRIF, we have analyzed its effects on second messenger activation in separate cultures of these subpopulations and have also evaluated the relative contribution of the corresponding signaling systems to the SRIF-stimulated GH release in these cells.

The second messenger pathways analyzed here were selected for their importance in the signaling mechanisms operating in pig somatotropes (37, 38, 41, 42) and those mediating SRIF effects in pituitary and nonpituitary cells (15, 16, 17, 40). Accordingly, we first studied the possible involvement of the AC/cAMP pathway in the stimulatory response of pig GH cells to SRIF by directly quantifying cAMP levels. This revealed a neat parallelism between the capacity of both SRIF doses (10-7 and 10-15 M) to augment cAMP content and GH release in the HD subpopulation that suggested a functional coupling between both events. However, the fact that such correlation was less apparent for the effects of a low SRIF dose on cAMP and GH release in LD cells together with our observation that LD and HD cell subpopulations are enriched in somatotropes but contain other hormonal cell types (13, 35) that probably contain SSTRs (16) raised the possibility that the SRIF-induced cAMP increase could represent a response of non-GH cells. Importantly, the use of an AC inhibitor excluded this latter possibility by showing that blockade of the pathway triggered by cAMP at an early step (AC activation) causes the complete loss of SRIF-induced GH secretion regardless of the dose of SRIF or subpopulation considered. Therefore, our present results unequivocally demonstrate the requisite role of the AC/cAMP route for the in vitro stimulatory action of SRIF on porcine GH release.

A phenomenon similar to the one described herein has not been reported previously in somatotropes or other pituitary cells. However, studies in nonpituitary cells and tissues have indicated that SRIF can indeed elevate intracellular cAMP levels. Specifically, high SRIF concentrations increased cAMP levels in tissues from two rat brain regions (43). More recently, transfection studies with cloned SSTRs from different species in CHO-K1 cells revealed that human SSTR5 and SSTR2 can both stimulate and inhibit AC in response to SRIF through a dose-dependent mechanism that involves different G proteins and is not reproducible using the same SSTRs from rat or mouse or other human SSTRs (44). Even with the cautious interpretation required by results derived from receptor expression in heterologous systems, these data clearly suggest that the stimulatory effect of SRIF is strongly species and receptor subtype specific. In line with this idea, the stimulation induced by SRIF on cAMP production and the subsequent GH release found here appear to be species specific.

The changes induced by SRIF in IP turnover in cultured LD and HD cells followed a pattern similar to that found for cAMP. However, this was less surprising, given the reported ability of SRIF to activate the PLC/IP pathway in vitro in various areas of the rat brain (45, 46), pituitary homogenates (46), and SSTR-expressing cell lines (47, 48, 49, 50). It was thus conceivable that this pathway could be involved in the stimulatory action of SRIF in GH secretion. However, PLC inhibition did not alter SRIF-induced GH release, indicating that IP turnover increase does not contribute substantially to this SRIF action. These results cannot be attributed to a lack of effect of the PLC inhibitor, as in parallel experiments conducted in our laboratory U-73122 reduced PLC-mediated GH release induced by GHRH (38) and by PACAP38 (41). As mentioned earlier, the SRIF-induced increases in IP turnover could correspond to a putative response of nonsomatotropic pituitary cells bearing SSTRs or could occur in somatotropes, but be uncoupled from GH release and influence a different cell function.

The ability of SRIF to reduce [Ca2+]i in somatotropes and the mechanisms mediating this effect have been thoroughly studied (1, 21, 23, 24, 25, 26, 27, 28, 29, 30). In fact, a direct relationship between such a reduction and inhibition of basal GH release has been proposed for rat somatotropes (21, 24). In line with this, a high SRIF dose decreased [Ca2+]i, albeit moderately, in LD porcine somatotropes. However, inasmuch as high SRIF concentrations do not inhibit basal GH release from this cell subpopulation or from intact, nonseparated cultures of dispersed porcine pituitary cells (12), or in the animal in vivo (8, 9), it seems reasonable to suggest that a similar pattern of SRIF-induced [Ca2+]i reduction coupled to inhibition of basal GH secretion does not exist in pig somatotropes. Indeed, the uncoupling between Ca2+ and GH release in LD somatotropes is further supported by our observation that a low SRIF dose, which stimulates hormone secretion in this subpopulation, did not significantly modify [Ca2+]i in single LD somatotropes. In support of this idea, an elegant study by Yunker and Chang (51) has recently shown that the effects of SRIF on [Ca2+]i and on GH release in fish somatotropes may not be parallel, but be totally uncoupled.

On the other hand, both high and low doses of SRIF significantly increased [Ca2+]i in HD somatotropes. In fact, this is the first evidence that SRIF can rise [Ca2+]i in a normal somatotrope, although the increases observed are lower than those evoked by GHRH (37) or PACAP (42) in pig somatotropes. Nevertheless, the capacity of SRIF to elevate [Ca2+]i is not restricted to pig HD somatotropes, as this effect has also been reported in cells from human adenomas (52, 53), and in different cell lines transfected with distinct SSTRs (48, 49, 54). Given the generally accepted role of Ca2+ in the stimulus-secretion model, it was anticipated that increases in [Ca2+]i induced by SRIF in HD somatotropes would be coupled to the corresponding SRIF-induced GH release in this subpopulation. However, neither external Ca2+ entry nor Ca2+ mobilization from internal stores was required for the GH-releasing action of SRIF. Again, these results cannot be explained by an ineffectiveness of the approach or the Ca2+ blockers used, which have been successfully employed in comparable experimental settings (38). Hence, these and our previous results comprise a compelling argument to propose that SRIF stimulates GH release from porcine LD somatotropes (at low doses) and HD somatotropes (at both high and low doses), through a mechanism that requires AC-mediated cAMP production, but seems to be independent of net increases in [Ca2+]i or increased IP turnover. The molecular scenario that mediates this SRIF action might not be readily apparent, given the accepted paradigm of [Ca2+]i increase-secretion coupling in endocrine cells (27, 28). However, a growing number of studies is showing that an increase in secretory activity can occur in the absence of net variations in [Ca2+]i in both neurons and endocrine cells (55). In particular, neurotransmitter release from rat hippocampal (56) and cerebellar (57) cells as well as insulin secretion from pancreatic ß-cells (58, 59, 60) can occur through a mechanism independent of a net [Ca2+]i increase and, quite interestingly, dependent on AC and PKA activation. Moreover, Ämmälä and co-workers (58) suggest that the cAMP system sensitizes the secretory machinery to Ca2+, i.e. that cAMP initiates exocytosis at a Ca2+ concentration that by itself does not elicit secretion. In view of this and our present findings it is not unreasonable to propose that a similar situation might occur in SRIF-stimulated pig GH secretion. Future studies should explore the precise mechanisms employed by cAMP and its downstream mediators (e.g. PKA-induced phosphorylations) to activate the exocytotic process in pig somatotropes.

The differential response induced by SRIF on LD and HD somatotropes in terms of both GH secretion and signaling is noteworthy and might help in elucidating the precise mechanisms underlying these SRIF actions. Given the reported ability of each SSTR subtype to activate distinct signaling pathways (15, 16, 17), we propose that the differential response of LD and HD cells to SRIF can be due to a dissimilar distribution of SSTR subtypes in these somatotrope subpopulations and/or to the signaling systems that each SSTR can activate therein. Unfortunately, only the porcine SSTR2 has been cloned hitherto and has not been thoroughly characterized (61). Nevertheless, inasmuch as none of the individual SSTR1–5 cloned to date in other species seems to fully mimic the signaling events induced by SRIF in pig somatotropes (15, 16, 17), it is conceivable that such signaling properties are mediated through a pig-specific SSTR. Alternatively, the recently reported ability of SSTRs to form heterodimers with distinct signaling capacities from their single SSTR components offers an additional scenario, in which a distinct balance of SSTR subtypes in LD and HD somatotropes would enable different SSTR heterodimers and thus different signaling responses to SRIF (62, 63). Finally, it is also plausible that a distinct distribution of the signaling pathway components in LD and HD somatotropes could contribute to their differential response to SRIF, as it has been reported that SRIF can activate, even through the same SSTR, multiple and even opposite intracellular signaling mechanisms (44, 50, 64, 65). Studies are underway in our laboratory aimed at identifying the SSTRs present in each subpopulation that will help to differentiate between these possibilities.

In conclusion, our present study demonstrates that SRIF can function as a true GH-releasing factor in cultures of porcine pituitary cells through a mechanism dependent upon AC/cAMP and apparently independent of net increases in IP and [Ca+2]i. These novel actions challenge classic views of SRIF as a mere inhibitor for somatotropes and suggest that it may exert a more complex, dual function in the control of swine GH release, wherein molecular heterogeneity of somatotropes would play a critical role. Indeed, the present results confirm and expand our previous findings on the morphological and functional heterogeneity of pig somatotropes by unveiling the unusual molecular signaling mechanisms underlying the ability of SRIF to evoke distinct responses in LD and HD somatotropes. Furthermore, these findings may also provide new insights to understand the heterogeneous response of pituitary tumors to treatment with SRIF analogs. Certainly, although this dual action of SRIF is novel for normal somatotropes, a similar capacity for SRIF to cause opposite responses has been reported in cells from pituitary tumors and in other cell types, particularly neurons (44, 45, 46, 48, 49, 50, 52, 53, 54, 66, 67). Hence, the study of cellular heterogeneity appears as a useful tool to clarify the roles and mechanisms of SRIF in controlling normal and abnormal somatotrope function. Further studies in porcine SRIF receptor expression and their coupling to intracellular signaling systems would contribute to elucidate the challenging questions raised by the present work.


   Acknowledgments
 
This work is dedicated to the inspiring memory of our colleague and friend L. Stephen Frawley. We thank Dr. A. F. Parlow, Pituitary Hormones and Antisera Center, Harbor-University of California-Los Angeles Medical Center, and Drs. D. J. Bolt and D. W. Caldwell, USDA Animal Hormone Program, Beltsville Agriculture Research Center, for the generous gift of pGH.


   Footnotes
 
This work was supported by Grants CVI-0139 (Plan Andaluz de Investigación, Junta de Andalucía, Spain) and IFD97-0582 and PB97-0454 (Ministerio de Educación y Cultura, Spain).

1 Present address: Fraser Laboratories, Department of Medicine, McGill University and Royal Victoria Hospital, Montréal, Québec, Canada H3A 1A1. Back

2 Present address: Department of Environmental Biology and Public Health, Faculty of Experimental Sciences, University of Huelva, Campus El Carmen, 21071 Huelva, Spain. Back

Abbreviations: AC, Adenylate cyclase; [Ca2+]i, free cytosolic calcium concentration; EIA, enzyme immunoassay; HD, high density; IBMX, 3-isobutyl-1-methylxanthine; LD, low density; pGH, porcine GH; PIP, phosphoinositide; SRIF, somatostatin; VSCC, voltage-sensitive Ca2+ channel.

Received August 9, 2001.

Accepted for publication November 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Frohman LA, Downs TR, Chomczynski P 1992 Regulation of growth hormone secretion. Front Neuroendocrinol 13:344–405[Medline]
  2. Giustina A, Veldhuis JD 1998 Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human. Endocr Rev 19:717–797[Abstract/Free Full Text]
  3. McMahon CD, Radcliff RP, Lookingland KJ, Tucker HA 2001 Neuroregulation of growth hormone secretion in domestic animals. Dom Anim Endocrinol 20:65–68[CrossRef][Medline]
  4. Frohman LA, Downs TR, Clarke IJ, Thomas GB 1990 Measurement of growth hormone-releasing factor and somatostatin in hypothalamic-portal plasma of unanesthetized sheep. Spontaneous secretion and response to insulin-induced hypoglycemia. J Clin Invest 86:17–24
  5. Drisko JE, Faidley TD, Chang CH, Zhang D, Nicolich S, Hora DF, McNamara L, Rickes E, Abribat T, Smith RG, Hickey GJ 1998 Hypophyseal-portal concentrations of growth hormone-releasing factor and somatostatin in conscious pigs: relationship to production of spontaneous growth hormone pulses. Proc Soc Exp Biol Med 217:188–196[Abstract]
  6. Roelfsema F, Biermasz NR, Veldman RG, Veldhuis JD, Frolich M, Stokvis-Brantsma WH, Wit JM 2001 Growth hormone (GH) secretion in patients with an inactivating defect of the GH-releasing hormone (GHRH) receptor is pulsatile: evidence for a role for non-GHRH inputs into the generation of GH pulses. J Clin Endocrinol Metab 86:2459–2464[Abstract/Free Full Text]
  7. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660[CrossRef][Medline]
  8. Farmer C, Randall G, Brazeau P 1992 In vivo growth hormone (GH) response to human GH-releasing factor (GRF) or somatostatin (SRIF) in foetal pigs. J Dev Physiol 17:93–97[Medline]
  9. Anderson LL, Ford JJ, Klindt J, Molina JR, Vale WW, Rivier J 1991 Growth hormone and prolactin secretion in hypophysial stalk-transected pigs as affected by growth hormone and prolactin-releasing and inhibiting factors. Proc Soc Exp Biol Med 196:194–202[Abstract]
  10. Torronteras R, Gracia-Navarro F, Elsaesser F 1996 Different effects of somatostatin on in vitro growth hormone release in two porcine breeds with different growth rates. J Neuroendocrinol 8:891–900[CrossRef][Medline]
  11. Torronteras R, Gracia-Navarro F, Elsaesser F 1997 Control of growth hormone secretion from porcine fetal and neonatal pituitary tissue in vitro by growth hormone-releasing hormone, somatostatin, and insulin-like growth factor. Neuroendocrinology 65:117–128[Medline]
  12. Ramírez JL, Castaño JP, Gracia-Navarro F 1998 Somatostatin at low doses stimulates growth hormone release from intact cultures of porcine pituitary cells. Horm Metab Res 30:175–177[Medline]
  13. Castaño JP, Torronteras R, Ramírez JL, Gribouval A, Sánchez-Hormigo A, Ruiz-Navarro A, Gracia-Navarro F 1996 Somatostatin increases growth hormone (GH) secretion in a subpopulation of porcine somatotropes: evidence for functional and morphological heterogeneity among porcine GH-producing cells. Endocrinology 137:129–136[Abstract]
  14. Ramírez JL, Torronteras T, Castaño JP, Sánchez-Hormigo A, Garrido JC, García-Navarro S, Gracia-Navarro F 1997 Somatostatin plays a dual, stimulatory/inhibitory role in the control of growth hormone secretion by two somatotrope subpopulations from porcine pituitary. J Neuroendocrinol 9:841–848[CrossRef][Medline]
  15. Reisine T, Bell GI 1995 Molecular biology of somatostatin receptors. Endocr Rev 16:427–443[CrossRef][Medline]
  16. Patel YC 1999 Somatostatin and its receptor family. Front Neuroendocrinol 20:157–198[CrossRef][Medline]
  17. Csaba Z, Dournaud P 2001 Cellular biology of somatostatin receptors. Neuropeptides 35:1–23[CrossRef][Medline]
  18. Kumar U, Laird D, Srikant CB, Escher E, Patel YC 1997 Expression of the five somatostatin receptor (SSTR1–5) subtypes in rat pituitary somatotrophes: quantitative analysis by double-layer immunofluorescence confocal microscopy. Endocrinology 138:4473–4476[Abstract/Free Full Text]
  19. Siehler S, Hoyer D 1999 Characterisation of human recombinant somatostatin receptors. III. Modulation of adenylate cyclase activity. Naunyn Schmiedebergs Arch Pharmacol 360:510–521[CrossRef][Medline]
  20. Epelbaum J, Enjalbert A, Krantic S, Musset F, Bertrand P, Rasolonjanahary R, Shu C, Kordon C 1987 Somatostatin receptors on pituitary somatotrophs, thyrotrophs and lactotrophs: pharmacological evidence for loose coupling to adenylate cyclase. Endocrinology 121:2177–2185[Abstract]
  21. Lussier BT, Wood DA, French MB, Moor BC, Kraicer J 1991 Free intracellular Ca2+ concentration ([Ca2+]i) and growth hormone release from purified rat somatotrophs. II. Somatostatin lowers [Ca2+]i by inhibiting Ca2+ influx. Endocrinology 128:583–591[Abstract]
  22. Musset F, Bertrand P, Kordon C, Enjalbert A 1990 Differential coupling with pertussis toxin-sensitive G proteins of dopamine and somatostatin receptors involved in regulation of adenohypophyseal secretion. Mol Cel Endocrinol 73:1–10[CrossRef][Medline]
  23. Goodyer CG, Branchaud CL, Lefebvre Y 1993 In vitro modulation of growth hormone (GH) secretion from early to midgestation human fetal pituitaries by GH-releasing factor and somatostatin. Role of Gs-adenylate cyclase-Gi complex and Ca2+ channels. J Clin Endocrinol Metab 76:1265–1270[Abstract]
  24. Holl RW, Thorner MO, Mandell GL, Sullivan JA, Sinha YN, Leong DA 1988 Spontaneous oscillations of intracellular calcium and growth hormone secretion. J Biol Chem 263:9682–9685[Abstract/Free Full Text]
  25. Holl RW, Thorner MO, Leong DA 1988 Intracellular calcium concentration and growth hormone secretion in individual somatotropes: effects of growth hormone-releasing factor and somatostatin. Endocrinology 122:2927–2932[Abstract]
  26. Rawlings SR, Hoyland J, Mason WT 1991 Calcium homeostasis in bovine somatotrophs: calcium oscillations and calcium regulation by growth hormone-releasing hormone and somatostatin. Cell Calcium 12:403–414[CrossRef][Medline]
  27. Lussier BT, French MB, Moor BC, Kraicer J 1991 Free intracellular Ca2+ concentration ([Ca2+]i) and growth hormone (GH) release from purified rat somatotrophs. III. Mechanism of action of GH-releasing factor and somatostatin. Endocrinology 128:592–603[Abstract]
  28. Chen C, Vincent JD, Clarke IJ 1994 Ion channels and the signal transduction pathways in the regulation of growth hormone secretion. Trends Endocrinol Metab 5:227–233[Medline]
  29. White RE, Schonbrunn A, Armstrong DL 1991 Somatostatin stimulates Ca(2+)-activated K+ channels through protein dephosphorylation. Nature 351:570–573[CrossRef][Medline]
  30. Kato M 1995 Withdrawal of somatostatin augments L-type Ca2+ current in primary cultured rat somatotrophs. J Neuroendocrinol 7:855–859[CrossRef][Medline]
  31. French MB, Moor BC, Lussier BT, Kraicer J 1989 Protein kinase C is not essential for growth hormone (GH)-releasing factor-induced GH release from rat somatotrophs. Endocrinology 124:2235–3344[Abstract]
  32. Cuttler L, Collins BJ, Szabo M 1995 Ontogeny of the GH response to phorbol ester and phospholipase C in rat pituitary cells. J Endocrinol 145:307–314[Abstract]
  33. Smith RG, Van der Ploeg LH, Howard AD, Feighner SD, Cheng K, Hickey GJ, Wyvratt Jr MJ, Fisher MH, Nargund RP, Patchett AA 1997 Peptidomimetic regulation of growth hormone secretion. Endocr Rev 18:621–645[Abstract/Free Full Text]
  34. Sánchez-Hormigo A, Castaño JP, Torronteras R, Malagón MM, Ramírez JL, Gracia-Navarro F 1998 Direct effects of growth hormone (GH)-releasing hexapeptide (GHRP-6) and GH-releasing factor (GRF) on GH secretion from cultured porcine somatotropes. Life Sci 63:2079–2088[CrossRef][Medline]
  35. Torronteras R, Castaño JP, Ruiz-Navarro A, Gracia-Navarro F 1993 Application of a Percoll density gradient to separate and enrich porcine pituitary cell types. J Neuroendocrinol 5:257–266[CrossRef][Medline]
  36. Castaño JP, Ruiz-Navarro A, Malagón MM, Hidalgo-Díaz C, Gracia-Navarro F 1997 Secretory and morphological heterogeneity of porcine somatotropes during postnatal development. J Neuroendocrinol 9:769–775[CrossRef][Medline]
  37. Ramírez JL, Torronteras R, Malagón MM, Castaño JP, García-Navarro S, González de Aguilar JL, Martínez-Fuentes AJ, Gracia-Navarro F 1998 Growth hormone-releasing factor (GRF) mobilizes cytosolic free calcium through different mechanisms in two somatotrope subpopulations from porcine pituitary. Cell Calcium 23:207–217[CrossRef][Medline]
  38. Ramírez JL, Castaño JP, Torronteras R, Martínez-Fuentes AJ, Frawley LS, García-Navarro S, Gracia-Navarro F 1999 Growth hormone (GH)-releasing factor differentially activates cyclic adenosine 3',5'-monophosphate- and inositol phosphate-dependent pathways to stimulate GH release in two porcine somatotrope subpopulations. Endocrinology 140:1752–1759[Abstract/Free Full Text]
  39. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
  40. Chadwick DJ, Cardew G, eds 1995 Somatostatin and its receptors. Ciba Found Symp 190. Chichester: Wiley & Sons
  41. Martínez-Fuentes AJ, Castaño JP, Gracia-Navarro F, Malagón MM 1998 Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 activate common and distinct intracellular signaling pathways to stimulate growth hormone secretion from porcine somatotropes. Endocrinology 139:5116–5124[Abstract/Free Full Text]
  42. Martínez-Fuentes AJ, Castaño JP, Malagón MM, Vázquez-Martínez R, Gracia-Navarro F 1998 Pituitary adenylate cyclase-activating polypeptides 38 and 27 increase cytosolic free Ca2+ concentration in porcine somatotropes through common and distinct mechanisms. Cell Calcium 23:369–378[CrossRef][Medline]
  43. Markstein R, Stöckli KA, Reubi JC 1989 Differential effects of somatostatin on adenylate cyclase as functional correlate for different brain somatostatin receptor subpopulations. Neurosci Lett 104:13–18[CrossRef][Medline]
  44. Carruthers AM, Warner AJ, Michel AD, Feniuk W, Humphrey PPA 1999 Activation of adenylate cyclase by human recombinant sst5 receptor expressed in CHO-K1 cells and involvement of G{alpha}s proteins. Br J Pharmacol 126:1221–1229[CrossRef][Medline]
  45. Muñoz-Acedo G, Izquierdo-Claros RM, Sánchez-Alonso JA, del Hoyo N, Pérez-Albarsanz MA, Arilla E 1995 Effect of somatostatin on the mass accumulation of inositol-1,4,5-trisphosphate in rat hypothalamus, striatum, frontoparietal cortex and hippocampus. Neurosci Lett 197:41–44[CrossRef][Medline]
  46. Lachowicz A, Pawlikowski M, Ochedalski T 1994 Somatostatin-14 increases the inositol-1,4,5-trisphosphate content in various areas of the brain. Biochem Biophys Res Commun 203:379–384[CrossRef][Medline]
  47. Wilkinson GF, Feniuk W, Humphrey PPA 1997 Characterization of human recombinant somatostatin sst5 receptors mediating activation of phosphoinositide metabolism. Br J Pharmacol 121:91–96[CrossRef][Medline]
  48. Akbar M, Okajima F, Tomura H, Majid MA, Yamada Y, Seino S, Kondo Y 1994 Phospholipase C activation and Ca2+ mobilization by cloned human somatostatin receptor subtypes 1–5, in transfected COS-7 cells. FEBS Lett 348:192–196[CrossRef][Medline]
  49. Siehler S, Hoyer D 1999 Characterisation of human recombinant somatostatin receptors. IV. Modulation of phospholipase C activity. Naunyn Schmiedebergs Arch Pharmacol 360:522–532[CrossRef][Medline]
  50. Chen LC, Fitzpatrick VD, Vandlen RL, Tashjian AH 1997 Both overlapping and distinct signaling pathways for somatostatin receptor subtypes SSTR1 and SSTR2 in pituitary cells. J Biol Chem 272:18666–18672[Abstract/Free Full Text]
  51. Yunker WK, Chang JP 2001 Somatostatin actions on a protein kinase C-dependent growth hormone secretagogue cascade. Mol Cell Endocrinol 175:193–204[CrossRef][Medline]
  52. Spada A, Reza-Elahi F, Lania A, Gil-del-Alamo P, Bassetti M, Faglia G 1991 Hypothalamic peptides modulate cytosolic free Ca2+ levels and adenylyl cyclase activity in human nonfunctioning pituitary adenomas. J Clin Endocrinol Metab 73:913–918[Abstract]
  53. Chen ZP, Xu S, Lightman SL, Hall L, Levy A 1997 Intracellular calcium ion response to somatostatin in cells from human somatotroph adenomas. Clin Endocrinol 46:45–53[CrossRef][Medline]
  54. Taylor JE 1995 Somatostatin (SSTR2) receptor mediates phospholipase C-independent Ca2+ mobilization in rat AR42J pancreas cells. Biochem Biophys Res Commun 214:81–85[CrossRef][Medline]
  55. Hille B, Billiard J, Babcock DF, Nguyen T, Koh DS 1999 Stimulation of exocytosis without a calcium signal. J Physiol 520:23–31[Abstract/Free Full Text]
  56. Wang HL, Li A, Wu T 1997 Vasoactive intestinal polypeptide enhances the GABAergic synaptic transmission in cultured hippocampal neurons. Brain Res 746:294–300[CrossRef][Medline]
  57. Kondo S, Mary A 1997 Protein-kinase A-mediated enhancement of miniature IPSC frequency by noradrenaline in rat cerebellar stellate cells. J Physiol 498:165–176[Medline]
  58. Ämmälä C, Ashcroft FM, Rorsman P 1993 Calcium-independent potentiation of insulin release by cyclic AMP in single ß-cells. Nature 363:356–358[CrossRef][Medline]
  59. Komatsu M, Shermerhorn T, Straub SG, Sharp GW 1996 Pituitary adenylate cyclase-activating peptide, carbachol, and glucose stimulate insulin release in the absence of an increase in intracellular Ca2+. Mol Pharmacol 50:1047–1054[Abstract]
  60. Ding WG, Gromada J 1997 Protein-kinase A-dependent stimulation of exocytosis in mouse pancreatic ß-cells by glucose-dependent insulinotropic polypeptide. Diabetes 46:615–621[Abstract]
  61. Matsumoto K, Yokogoshi Y, Fujinaka Y, Zhang C, Saito S 1994 Molecular cloning and sequencing of porcine somatostatin receptor 2. Biochem Biophys Res Commun 199:298–305[CrossRef][Medline]
  62. Rocheville M, Lange DC, Kumar U, Sasi R, Patel RC, Patel YC 2000 Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers. J Biol Chem 275:7862–7869[Abstract/Free Full Text]
  63. Pfeiffer M, Koch T, Schroder H, Klutzny M, Kirscht S, Kreienkamp HJ, Hollt V, Schulz S 2001 Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst3 receptor function by heterodimerization with sst2A. J Biol Chem 276:14027–14036[Abstract/Free Full Text]
  64. Murthy KS, Coy DH, Makhlouf GM 1996 Somatostatin receptor-mediated signaling in smooth muscle-activation of phospholipase Cß3 by Gß{gamma} and inhibition of adenylyl cyclase by G{alpha}i1 G{alpha}o. J Biol Chem 271:23458–23463[Abstract/Free Full Text]
  65. Douziech N, Calvo E, Coulombe Z, Muradia G, Bastien J, Aubin RA, Lajas A, Morisset J 1999 Inhibitory and stimulatory effects of somatostatin on two human pancreatic cancer cell lines: a primary role for tyrosine phosphatase SHP-1. Endocrinology 140:765–777[Abstract/Free Full Text]
  66. Delfs JR, Dichter MA 1983 Effects of somatostatin on mammalian cortical neurons in culture: physiological actions and unusual dose response characteristics. J Neurosci 3:1176–1188[Abstract]
  67. Lanneau C, Viollet C, Faivre-Bauman A, Loudes C, Kordon C, Epelbaum J, Gardette R 1998 Somatostatin receptor subtypes sst1 and sst2 elicit opposite effects on the response to glutamate of mouse hypothalamic neurones: an electrophysiological and single cell RT-PCR study. Eur J Neurosci 10:204–212[CrossRef][Medline]



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