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Endocrinology Vol. 140, No. 4 1752-1759
Copyright © 1999 by The Endocrine Society

ARTICLES

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 Subpopulations1

J. L. Ramírez, J. P. Castaño, R. Torronteras, A. J. Martínez-Fuentes, L. S. Frawley, S. García-Navarro and F. Gracia-Navarro

Department of Cell Biology, University of Córdoba (J.L.R., J.P.C., R.T., A.J.M.-F., S.G.-N., F.G.-N.), 14004 Córdoba, Spain; and the Department of Cell Biology and Anatomy, Medical University of South Carolina (L.S.F.), Charleston, South Carolina 29425

Address all correspondence and requests for reprints to: Dr. F. Gracia-Navarro, Department of Cell Biology, Faculty of Sciences, Avda. San Alberto Magno s/n, University of Córdoba, 14004 Córdoba, Spain. E-mail: bc1grnaf{at}uco.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Somatotropes comprise two morphologically and functionally distinct subpopulations of low (LD) and high (HD) density cells. We recently reported that GRF induces different patterns of increase in the cytosolic free Ca2+ concentration in single porcine LD and HD somatotropes, which for LD cells required not only Ca2+ influx but also intracellular Ca2+ mobilization. This suggested that GRF may activate multiple signaling pathways in pig LD and HD somatotropes to stimulate GH secretion. To address this question, we first assessed the direct GRF effect on second messenger activation in cultures of LD and HD cells by measuring cAMP levels and [3H]myo-inositol incorporation. Secondly, to determine the relative importance of cAMP- and inositol phosphate (IP)-dependent pathways, and of intra- and extracellular Ca2+, GRF-induced GH release from cultured LD and HD somatotropes was measured in the presence of specific blockers. GRF increased cAMP levels in both subpopulations, whereas it only augmented IP turnover in LD cells. Accordingly, adenylate cyclase inhibition by MDL-12,330A abolished GRF-stimulated GH release in both subpopulations, whereas phospholipase C inhibition by U-73122 only reduced this effect partially in LD cells. Likewise, blockade of Ca2+ influx with Cl2Co reduced GRF-stimulated GH secretion in both LD and HD somatotropes, whereas depletion of thapsigargin-sensitive intracellular Ca2+ stores only decreased the secretory response to GRF in LD cells. These results demonstrate that GRF specifically and differentially activates multiple signaling pathways in two somatotrope subpopulations to stimulate GH release. Thus, although the prevailing signaling cascade employed by GRF in both subpopulations is adenylate cyclase/cAMP/extracellular Ca2+, the peptide also requires activation of the phospholipase C/IP/intracellular Ca2+ pathway to exert its full effect in porcine LD somatotropes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HYPOTHALAMIC peptide GRF is the primary evocative stimulus for pituitary somatotropes (1, 2, 3) and exerts its actions on GH secretion and gene transcription through a specific membrane receptor(s) that belongs to the seven-transmembrane-spanning, G protein-linked family of receptors (4). Binding of GRF to its receptor activates at least two major signaling pathways that involve the second messengers cAMP and Ca2+, respectively (reviewed in Refs. 1, 3, 5). GRF increases cAMP levels by stimulating adenylate cyclase (AC) through a Gs protein (1, 3, 6, 7, 8). Subsequently, increased cAMP levels induce activation of protein kinase A (PKA), which, in turn, activates several proteins by phosphorylation, including ion channels (1, 3, 4, 9). Likewise, GRF increases the cytosolic free Ca2+ concentration ([Ca2+]i) in somatotropes from a number of species, including human (10), pig (11), rat (12, 13), cow (14), and sheep (15). Current information indicates that this rise in [Ca2+]i is essentially dependent on the entry of extracellular Ca2+ through voltage-sensitive Ca2+ channels (VSCC) (reviewed in Ref. 5). The precise mechanism(s) that activates such VSCCs has not been fully elucidated, but there is evidence for the involvement of Na+ channels and PKA-dependent phosphorylation(s) in this process (3, 5, 16).

Although cAMP production and Ca2+ influx constitute two pivotal mechanisms employed by GRF to exert its actions on somatotropes, it has been suggested that this peptide can also activate other signaling mechanisms in these cells (1, 3). Accordingly, results from a limited number of studies (17, 18; reviewed in Refs. 1, 3) have raised doubts against the prevailing view that GRF does not activate the phospholipase C (PLC)/inositol phosphate (IP) pathway and the subsequent mobilization of intracellular Ca2+ in rat somatotropes (12, 13, 19, 20, 21). That this system is present and functional in somatotropes has been clearly demonstrated by studies on the mechanisms of action of synthetic GH secretagogues such as GH-releasing hexapeptide (GHRP-6) (22, 23, 24). However, whereas earlier reports showed GRF to stimulate phosphatidyl inositol turnover in cultured rat pituitary cells (17), other researchers later reported that GRF did not significantly alter 32P incorporation into IPs in purified rat somatotropes (20). Similarly, Ohlsson and Lindström (18) demonstrated that GRF stimulated Ca2+ efflux from purified somatotropes in a Ca2+-depleted medium and thereby suggested that it mobilized Ca2+ from intracellular pools. However, other studies have reported that both the GH release and the [Ca2+]i increase stimulated by GRF in purified somatotropes were inhibited when somatotropes were incubated in Ca2+-free medium or treated with a Ca2+ antagonist (12). Therefore, there are still some aspects of the possible involvement of the PLC/IP/intracellular Ca2+ system in the response of somatotropes to GRF that require clarification (1, 3, 21).

In the present study, we have investigated the relative contributions of different intracellular signaling pathways to the secretory response of porcine somatotropes to GRF. The reason for selecting this particular model is 2-fold: first, because the mechanisms employed by GRF to release GH in vitro in this species have not been addressed hitherto, and second, and more important, because recent findings on the functional heterogeneity of pig somatotropes suggested a possible cellular basis for the complex response of this cell type to GRF (11). Indeed, previous studies from our group showed that the porcine somatotrope population is heterogeneous and can be separated by density gradient centrifugation into two major subpopulations of low (LD) and high (HD) density cells, which display substantial differences in ultrastructure, secretory capacity, and response to regulatory factors (25, 26, 27). In particular, GRF was recently found to induce a differential response in [Ca2+]i dynamics in LD and HD somatotropes. Thus, the response of LD cells to GRF depended on both extracellular Ca2+ influx and mobilization of Ca2+ from intracellular stores, whereas that of HD somatotropes was exclusively dependent on extracellular Ca2+ entry through L-type VSCCs (11). Inasmuch as these results suggested that GRF may differentially activate distinct signaling cascades in each subpopulation, the aim of the present study was to elucidate which pathways underlie the secretory response of porcine LD and HD somatotropes to GRF. To answer this question, cAMP levels and [3H]myo-inositol incorporation were quantified in cultures of separated subpopulations after GRF treatment. Furthermore, specific inhibitors of distinct signaling pathways were employed to ascertain the relative importance of such routes to GRF-stimulated GH secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, cell dispersion, and separation of subpopulations
Prepubertal Large-White/Landrace female pigs (5–6 months old) were obtained from a local slaughterhouse. Animals were killed by exsanguination after brief electrical stunning, and pituitary glands were immediately removed and transferred to sterile cold (4 C) medium (D-valine-modified MEM; Sigma Chemical Co., London, UK) supplemented with 0.1% BSA (Sigma Chemical Co.) and antibiotic-antimycotic solution (Sigma Chemical Co.). In the laboratory, pituitaries were washed twice with fresh medium, and the posterior lobes were discarded. For each separate, independent experiment, six to eight pituitary glands were pooled and dispersed together by means of an enzymatic-mechanical dispersion method described previously (25, 26). Briefly, glands were cut into fragments of 1–2 mm3 and then exposed to sequential incubation with 0.3% trypsin (type I, Sigma Chemical Co.), 0.1% collagenase (type V, Sigma Chemical Co.), 0.1% soybean trypsin inhibitor I (Sigma Chemical Co.), 2 µg/ml deoxyribonuclease (Sigma Chemical Co.), and Ca2+/Mg2+-free HBSS (Sigma Chemical Co.) with EDTA (2 and 1 mM), followed by mechanical dispersion with a Pasteur pipette. Cellular viability, as determined by the trypan blue exclusion test, was always higher than 85%.

Separation of somatotrope subpopulations was carried out by centrifugation in a Percoll density gradient as previously reported (25, 26). In short, 30–40 x 106 dispersed cells were resuspended in 0.5 ml medium and carefully loaded on top of a hyperbolic, continuous Percoll density gradient (Pharmacia LKB, Uppsala, Sweden). This preparation was centrifuged at 3000 x g for 25 min, and two somatotrope subpopulations of LD (1.051–1.064 g/ml) and HD (1.087–1.098 g/ml) were isolated, which have been thoroughly characterized in previous studies (26, 27). Dispersed cells from each subpopulation were cultured in MEM supplemented with 10% FBS (Sera-Lab, Crawley-Down, UK) in a humidified atmosphere of 5% CO2-95% air at 37 C. Experiments were carried out after 4 days of culture. A 2-h preincubation in fresh MEM without FBS was used to stabilize basal GH secretion before adding test substances.

Measurement of cAMP
To measure cAMP, LD and HD cell subpopulations were plated in six-well tissue culture plates at a density of 2 x 106 cells/well·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 Chemical Co.) 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 10-8 M GRF-(1–29) (UCB Bioproducts, Brain L’Alleud, Belgium). Medium was then aspirated, and wells were scraped in 0.01 M PBS with 4 mM EDTA. Subsequently, aliquots for protein determination were removed, and samples were sonicated (5 min), boiled (10 min), and centrifuged at 10,000 rpm for 5 min. The supernatant was removed and stored at -20 C until cAMP determination by means of a [3H]cAMP assay kit (Amersham, Aylesbury, UK). Results are reported as picomoles of cAMP per mg total protein.

Measurement of [3H]myo-inositol incorporation
To evaluate [3H]myo-inositol incorporation, LD and HD cells were plated in 12-well plates at 6 x 105 cells/well·2 ml MEM-FBS. After the 2-h preincubation in FBS-free MEM, cells were incubated for 30 min in 500 µl MEM containing [3H]myo-inositol (3 µCi/ml; 10–25 Ci/mmol; DuPont, Stevenage, UK) in the presence or absence of 10-8 M GRF. 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 in 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 then added to the pellet to extract the phosphoinosi-tides (PIPs). After centrifugation (12,000 rpm, 20 min), the supernatant was also removed and measured. Because 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 the total amount of [3H]myo-inositol incorporated in each sample (IPs plus PIPs) and are expressed as counts per min/mg total protein.

Evaluation of GH secretion
In this set of experiments, dispersed cells from each subpopulation were cultured during 4 days at a density of 3 x 105/well/ml in 24-well plates. After the 2-h preincubation in FBS-free MEM, cells were incubated for an additional 2-h period with MEM alone or MEM that contained one of the following inhibitors: 1) AC inhibitor, 10 µM MDL-12,330A (Research Biochemicals International, Natick, MA) (28); 2) PLC inhibitor, 50 µM U-73122 (Research Biochemicals International) (29, 30); 3) VSCC blocker, 2 mM CoCl2 (14, 31); and 4) sarco/endoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor (to deplete intracellular Ca2+ stores), 100 nM thapsigargin (TG; Sigma Chemical Co.) (11, 22, 32). Immediately thereafter, cells were challenged for 30 min with 10-8 M GRF in the presence or absence of the corresponding inhibitor. Then, medium was removed and microfuged, and the supernatant was stored at -20 C until porcine GH was measured by means of a homologous enzyme immunoassay (EIA), described previously (26). Hormone employed in the EIA for both plate coating and standard was pGH (USDA-B-1, AFP-11716C; kindly supplied by Dr. A. Parlow, Pituitary Hormones and Antisera Center, Harbor University of California-Los Angeles Medical Center, Los Angeles, CA), and the primary antiserum was a specific anti-pGH (raised in rabbit; UCB) at a dilution of 1:200,000. The sensitivity of the EIA was 0.65 ± 0.12 ng pGH/well.

Statistical analysis
Data are expressed as the mean ± SEM obtained from at least three separate, independent experiments. In each experiment, at least four replicate wells were tested per treatment group. Statistical analysis was performed with 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 GRF-stimulated GH release
Measurement of cAMP in cultures of separated subpopulations of porcine pituitary cells demonstrated that cAMP levels were higher in LD than in HD cells under basal culture conditions (14.1 ± 2.0 vs. 6.6 ± 1.9 pmol/mg protein, respectively; Fig. 1Go). In both subpopulations, GRF (10-8 M; 30 min) evoked a significant increase in cAMP levels (Fig. 1Go), that was proportionally higher for HD (3-fold; lower panel) than for LD cells (2-fold; upper panel). To examine the possible contribution of this cAMP rise to GRF-induced GH release from LD and HD somatotropes, we evaluated the effect of the specific AC inhibitor MDL-13,330A on the stimulation caused by the peptide. None of the inhibitors used in this and the remaining experiments that analyzed other intracellular signaling pathways significantly altered basal GH secretion from either subpopulation. However, as indicated by the results depicted in Fig. 2Go, MDL-12,330A abolished the stimulatory effect of GRF in both LD (upper panel), and HD (lower panel) cells. These results demonstrate that activation of the AC-cAMP system is required by GRF to stimulate the secretory response of both porcine somatotrope subpopulations.



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  		Figure 1. Effect of GRF on cAMP production in monolayer cultures of porcine LD (upper panel) and HD (lower panel) pituitary cells. After 4 days of culture in MEM-FBS, cells were equilibrated for 2 h in serum-free MEM and then incubated with 1 mM IBMX for 30 min. After incubation, cells were challenged for 30 min with 10-8 M GRF in presence of IBMX. 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 GRF-stimulated GH secretion in LD (upper panel) and HD (lower panel) porcine somatotropes. After 4 days 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. Then, cells were challenged with 10-8 M GRF for 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 percentage of the respective control value (100%; dotted line). a, P < 0.05 vs. corresponding control; b, P < 0.05 vs. GRF alone.

 
Role of the IP-dependent pathway in GRF-stimulated GH release
Similar to that found for cAMP, levels of [3H]myo-inositol incorporation under basal culture conditions were higher for LD than for HD cells (4.5 ± 1.2 vs. 2.5 ± 0.8 cpm/mg protein, respectively; Fig. 3Go). In this case, GRF (10-8 M; 30 min) only elicited a significant increase in [3H]myo-inositol incorporation in LD cells (3-fold over the control value; Fig. 3Go, upper panel), whereas it did not affect [3H]myo-inositol uptake in HD cells under the same conditions (Fig. 3Go, lower panel). To ascertain the possible role of the PLC-IP pathway in the secretory response of each subpopulation to GRF, we employed the specific PLC inhibitor, U-73122. The presence of this agent caused a significant, albeit partial, decrease in GRF-stimulated GH release in LD cells (Fig. 4Go, upper panel), whereas it did not affect the stimulation induced by GRF in HD somatotropes (Fig. 4Go, lower panel). Therefore, normal activation of the PLC-IP pathway seems to be requisite for GRF to fully stimulate GH release only in LD somatotropes, but not in HD cells.



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  		Figure 3. Incorporation of [3H]myo-inositol in monolayer cultures of porcine LD (upper panel) and HD (lower panel) cells in response to GRF. After 4 days of culture in MEM-FBS, cells were equilibrated for 2 h in serum-free MEM and then incubated with 10-8 M GRF for 30 min in the presence of [3H]myo-inositol. Thereafter, samples were recovered, and labeled IPs and PIPs were measured. 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. control.

 


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  		Figure 4. Secretory response of LD (upper panel) and HD (lower panel) porcine somatotropes to 10-8 M GRF after blockade of PLC activity. After 4 days 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 10-8 M GRF 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; b, P < 0.05 vs. GRF alone.

 
Role of the extra- and intracellular Ca2+in GRF-stimulated GH release
The relative importance of extracellular Ca2+ entry into somatotropes on GRF-induced GH release was assessed using CoCl2, a blocker of VSCCs. As shown in Fig. 5Go, addition of CoCl2 to the incubation medium strongly reduced GRF-stimulated GH release in both subpopulations. In contrast, depletion of intracellular Ca2+ stores with TG, a sarco/endoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor, partially reduced the stimulatory effect of GRF only in LD somatotropes (159.3 ± 5.5% of the control value; Fig. 6Go, upper panel), without affecting the increase in GH release induced by this peptide in HD cells (Fig. 6Go, lower panel). These results indicate that extracellular Ca2+ plays a critical role in the secretory response of both LD and HD porcine somatotropes to GRF, whereas Ca2+ mobilization from intracellular stores seems to participate only in the secretory response of LD somatotropes to GRF.



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  		Figure 5. Effect of blockade of Ca2+ influx by 2 mM CoCl2 on GRF-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; b, P < 0.05 vs. GRF alone.

 


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  		Figure 6. Effect of depletion of intracellular Ca2+ stores by 100 nM TG on GRF-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; b, P < 0.05 vs. GRF alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies from our laboratory have shown that the population of porcine somatotropes is composed of two major subpopulations (LD and HD somatotropes) that can be separated by centrifugation in a density gradient and that exhibit ultrastructural and functional differences (25, 26, 27, 33). Recently, analysis of the GRF-induced [Ca2+]i responses of LD and HD somatotropes demonstrated the existence of marked differences between the two supopulations in the profiles of the [Ca2+]i rise induced by the peptide as well as in the source of Ca2+ accounting for such elevations (11). In fact, whereas GRF-stimulated Ca2+ response in LD somatotropes was dependent on Ca2+ from both intra- and extracellular origins, the response of HD somatotropes was only dependent on extracellular Ca2+ entry (11). These findings suggested a differential activation of intracellular signaling pathways by GRF in both subpopulations. Accordingly, in the present study we found that stimulation of GH release by GRF in LD somatotropes is exerted through activation of two different second messenger systems, namely AC/cAMP/extracellular Ca2+ and PLC/IP/intracellular Ca2+, whereas only the former pathway is involved in the secretory response of HD somatotropes.

A first approach toward evaluating the possible involvement of different signaling pathways in the response of porcine somatotropes to GRF was to directly determine cAMP levels and IP turnover in cultures of LD and HD cells. Interestingly, initial results revealed clear differences in basal levels of second messengers in these subpopulations, in that both cAMP levels and [3H]myo-inositol incorporation were higher in LD than in HD cells. As both subpopulations are enriched in somatotropes (i.e. composed of at least 40–50% GH cells) (26), it is likely that such differences in cAMP and IP may be attributed at least in part to somatotropes within each subpopulation. These results would be consistent with our previous findings that these same subpopulations exhibited comparable differences in basal [Ca2+]i levels measured at the single somatotrope level (11). Moreover, the reduced rate of second messenger activity detected in the HD subpopulation may help to explain the observation that these cells contain 4-fold more GH, but only release twice the amount of hormone as LD somatotropes (26). Thus, taken together, these and previous results suggest that heterogeneity of porcine LD and HD somatotropes includes not only the reported differences in density, ultrastructure, and response to regulatory factors, but also molecular differences in terms of second messenger levels under basal culture conditions.

It is widely accepted that two major events in the response of somatotropes to GRF are a cAMP increase and extracellular Ca2+ entry (1, 3, 5, 21). Consistent with this idea, our results indicated the critical role played by the AC/cAMP/extracellular Ca2+ signaling system in the stimulatory action of GRF in both porcine somatotrope subpopulations. Thus, the ability of GRF to increase cAMP levels coupled with the observation that GRF-stimulated GH release was blocked in the presence of an AC inhibitor demonstrated the requisite participation of the AC/cAMP system in mediating the effect of the peptide in both LD and HD somatotropes. Likewise, the decrease in GRF-induced GH release observed when VSCCs were blocked with CoCl2 indicated clearly the importance of extracellular Ca2+ entry for the stimulatory effect of GRF. These latter results confirm and expand our previous observations that Ca2+ from extracellular origin is involved in the GRF-induced [Ca2+]i elevation in both somatotrope subpopulations (11). The precise sequence of events linking GRF activation of the AC/cAMP/PKA pathway, external Ca2+ entry, and hormone release has not been fully elucidated. In rat and human somatotropes, it has been reported that PKA-dependent phosphorylation of Ca2+, Na+, and nonselective cation channels and/or related proteins (1, 3, 5, 16, 34) may participate in this complex mechanism. In porcine somatotropes, it has been recently shown that inactivation of PKA by H-89 drastically reduced the effect of pituitary adenylate cyclase-activating polypeptide on extracellular Ca2+ entry through VSCC, thereby indicating that a PKA-dependent phosphorylation of such channels or related proteins would precede Ca2+ entry (35). It is thus conceivable that a similar mechanism operates in the case of GRF in porcine somatotropes. Nevertheless, it is noteworthy that whereas AC inhibition abolished the effect of GRF on GH release, blockade of Ca2+ by CoCl2 partially reduced, but did not abolish, GRF stimulation. As in porcine somatotropes CoCl2 has been shown to prevent [Ca2+]i increases mediated by Ca2+ influx (11), the present results raise the interesting possibility that activation of the AC/cAMP/PKA cascade in these cells induces effects on GH release that are independent of Ca2+ entry. In support of this idea, recent studies on insulin-secreting mouse ß-cells revealed a mechanism by which cAMP induced exocytosis in a manner independent of net increases in [Ca2+]i (36, 37). This might represent an additional mechanism for cAMP to potentiate GH release in somatotropes, as has been suggested for human GH-releasing adenoma cells (34).

The possible involvement of the PLC/IP pathway and of intracellular Ca2+ stores in the stimulatory action of GRF on GH secretion has been a matter of controversy in recent years. Although some researchers argue that such signaling mechanisms are not involved in this response, others have provided evidence suggesting their participation (1, 3, 5, 12, 13, 17, 18, 19, 20, 21). Our strategy of studying separated, functionally distinct subpopulations of somatotropes may shed light on this issue. Accordingly, GRF increased IP turnover in cultures of LD, but not HD, cells, and inhibition of PLC partially reduced GRF-stimulated GH secretion from LD somatotropes without altering that from companion cultures of HD cells. Likewise, mobilization of Ca2+ from intracellular stores, a common subsequent step after PLC/IP pathway activation, was only required in the response of LD somatotropes, as depletion of these stores by thapsigargin blunted GRF-stimulated GH secretion in LD, but not in HD, somatotropes. Taken together, these results demonstrate the differential involvement of the PLC/IP/intracellular Ca2+ pathway in the secretory response of porcine LD and HD somatotropes to GRF. This idea was initially suggested by a previous study in which the GRF-evoked [Ca2+]i increase in single LD somatotropes was partly due to mobilization of Ca2+ from internal stores, whereas the rise of [Ca2+]i in HD somatotropes was exclusively dependent on extracellular Ca2+ entry. It is presently unknown whether this situation also occurs in somatotropes from other species. If that were the case, it is plausible that negative results from previous studies on the participation of the PLC/IP/intracellular Ca2+ pathway in the response to GRF could be attributed to the limited contribution of this signaling route in comparison with the clear predominance of the AC/cAMP/PKA pathway in this response. In other cases, the use of purified preparations of somatotropes may have selected specific subpopulations of this cell type in which the PLC/IP/intracellular Ca2+ pathway is not activated in response to GRF (12, 13, 20). Furthermore, given the dynamic changes experienced by LD and HD somatotrope subpopulations during postnatal development (27) and aging (38), it is possible that the differential involvement of IP and cAMP in the somatotrope response to GRF may vary with animal age. This consideration notwithstanding, the present data clearly demonstrate the existence of molecular heterogeneity in the response of porcine somatotrope subpopulations to GRF in terms of intracellular signaling mechanisms (Fig. 7Go), which may help in understanding the seemingly contradictory results found previously for this cell type.



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  		Figure 7. Schematic diagram summarizing the possible signaling pathways and mechanisms involved in the GRF-induced stimulatory response of porcine LD and HD somatotropes. GRF-R, GRF receptor; {alpha}ß{gamma}, subunits of heterotrimeric G proteins (Gs and Gp); IP3, inositol 1,4,5-trisphosphate; Ca2+, cytosolic free calcium. See Discussion for details.

 
The exact mechanisms underlying the molecular heterogeneity of the signaling cascades activated in LD and HD somatotropes in response to GRF await elucidation. However, two possible explanations should be considered that include both receptor and postreceptor events. In the first case, the differential activation of second messenger systems in somatotrope subpopulations might be related to the existence of different GRF receptors, which, in turn, could be coupled to different effector systems. Consistent with this idea, Hsiung et al. (39) isolated two GRF receptor complementary DNA forms from porcine pituitary that differed in the presence or absence of a 28-amino acid fragment. The shorter form of the receptor is predominant in the porcine pituitary gland and is identical to human, rat, and mouse cloned receptors, whereas the longer form does not yet have a counterpart in those species, although a similar rat GRF receptor has been described by Mayo et al. (4). It is conceivable that if such GRF receptor subtypes were functional in porcine somatotropes, one subtype could be predominant and activate the AC-cAMP pathway, whereas another receptor subtype, only expressed in LD cells, could activate the PLC-IP system. Thus, GRF receptor variants with a heterogeneous distribution in porcine somatotrope subpopulations could explain the differential activation of second messenger systems by GRF in these cells. However, it is also possible that a single type of receptor can activate both pathways, as has been reported to occur for the porcine receptor for PTH/PTH-related peptide. Indeed, when this receptor, which belongs to the same family as the GRF receptor, was expressed in the human kidney cell line 293, treatment with PTH increased both cAMP and IP production (40). If a similar multipotential GRF receptor were to exist in porcine somatotropes, the precise route(s) activated in each subpopulation would be determined by the molecular environment (G proteins, enzymes) present in the cells. Finally, there is an alternative, nonexcluding possibility, in which activation of the PLC/IP pathway in LD somatotropes could be exerted through an indirect mechanism. To be more specific, ß{gamma} complexes of heterotrimeric G proteins released after receptor activation have been shown to interact directly or indirectly with different effectors, including PLC (41, 42). In this scenario, the GRF receptor could activate AC through the {alpha}-subunit of G protein, whereas the ß{gamma} complex would activate PLC, as has been shown in human HL-60 granulocytes (43). In support of this latter possibility is our finding that the blocking of AC action abolished GH release in LD cells, thus suggesting that the partial contribution of the PLC/IP pathway to the GRF effect is dependent upon activation of the AC/cAMP system. Experiments underway in our laboratory are aimed at distinguishing among these possibilities.

In conclusion, our results reveal that GRF-induced GH release is mediated through differential activation of two intracellular signaling pathways in LD and HD subpopulations of porcine somatotropes. Although the AC/cAMP/extracellular Ca2+ system is the predominant route employed by GRF in both subpopulations, the peptide also requires activation of the PLC/IP/intracellular Ca2+ route to exert its full effect in LD somatotropes. Further studies will be required to ascertain the relevance of this somatotrope heterogeneity in second messenger activation and to elucidate the possible role of GRF receptor(s) in this phenomenon.


   Acknowledgments
 
We thank Dr. A. F. Parlow from the Pituitary Hormones and Antisera Center, Harbor-University of California-Los Angeles Medical Center, and Drs. D. J. Bolt and D. W. Caldwell, from USDA Animal Hormone Program, Beltsville Agriculture Research Center, for the generous gift of pGH.


   Footnotes
 
1 This work was supported by Grants CRG-971039 (NATO), CVI-0139 (Plan Andaluz de Investigación, Junta de Andalucía, Spain), and PB97–0454 (Ministerio de Educación y Cultura, Spain). Back

Received September 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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