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Department of Cell Biology, University of Córdoba, 14004-Córdoba, Spain
Address all correspondence and requests for reprints to: Dr. F. Gracia-Navarro, Department of Cell Biology. Faculty of Sciences, University of Córdoba, Avinguda San Alberto Magno, s/n, 14004-Cordoba, Spain. E-mail: bc1grnaf{at}uco.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PACAP exerts its actions through specific membrane receptors that belong to the seven transmembrane-spanning, G protein-linked family of receptors (1, 2, 19, 20). In rat, three major classes of PACAP/VIP receptors (PVRs), namely PAC1, VPAC1, and VPAC2 (previously known as PVR1, PVR2, and PVR3, respectively; 21), have been cloned and found to be expressed in the pituitary gland (1, 2, 19, 22, 23, 24, 25). PVRs can be distinguished by their binding affinities to PACAP38, PACAP27, and VIP, and by their ability to activate distinct signaling pathways (1, 2, 19). Furthermore, functional expression of five splice variants of the rat PAC1 has revealed that PACAP38 and PACAP27 can couple differentially to the adenylate cyclase (AC)/cAMP/protein kinase A (PKA) or the phospholipase C (PLC)/inositol phosphate (IP) pathways (22). In normal rat somatotropes, PACAP38 has been shown to stimulate Ca2+ influx through a cAMP-dependent mechanism (26, 27, 28), although the type of receptors and signaling pathways that mediate PACAP action in this cell type have not been fully elucidated. Furthermore, there is no evidence as to whether PACAP38 and PACAP27 couple to common or distinct signaling cascades in rat somatotropes. In contrast, we have recently reported that in porcine somatotropes both PACAP38 and PACAP27 increase cytosolic free Ca2+ concentration ([Ca2+]i) by stimulating extracellular Ca2+ entry through L-type VSCC by a PKA-dependent mechanism, but that PACAP38 also triggers a PLC-mediated Ca2+ mobilization from intracellular stores (29). These findings further suggested that the two molecular forms of PACAP could stimulate the secretory activity of porcine somatotropes by activating both distinct and common signaling pathways.
In the present study, we aimed at elucidating this question using cultures of porcine somatotropes to determine the second messengers that mediate the stimulatory effects of PACAP38 and PACAP27 on GH release and GH mRNA accumulation. To this end, hormone release and intracellular GH mRNA content were evaluated after PACAP treatment in combination with selective activators/inhibitors of the AC/cAMP/PKA and the PLC/IP/protein kinase C (PKC) pathways, as well as with blockers of extra- and intracellular Ca2+.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Drugs
Stock solutions of PACAP38 and PACAP27 (Peninsula Laboratories, Inc. Europe Ltd., Merseyside, UK), MDL 12,330 A
(Research Biochemicals International, Natick, MA), H89
(Calbiochem Corp., San Diego, CA), as well as dibutyril
cAMP (dbcAMP) and thapsigargin (Sigma Chemical Co.) were
prepared with distilled deionized water. U73122 (Research Biochemicals International), 1-O-tetradecanoyl
phorbol-13-acetate (TPA), and forskolin (Sigma Chemical Co.) were dissolved in dimethyl sulfoxide. Phloretin
(Calbiochem Corp.), nifedipine, and verapamil (Sigma Chemical Co.) were dissolved in ethanol. Aliquots of
concentrated stock solutions were stored at -20 C until use, when they
were diluted to final concentrations in MEM. The highest concentration
of dimethyl sulfoxide or ethanol was < 0.1%, which had no effect
on either basal hormone release or GH mRNA accumulation.
Animals and pituitary cell dispersion
Pituitaries from prepubertal Large White-Landrace sows (5–6
months old) were obtained from a local abattoir. Animals were killed by
exsanguination after electrical stunning. Within 5–10 min after death,
pituitaries were excised and transferred to sterile cold (4 C) medium
(MEM) supplemented with 0.1% BSA (fraction V). In the laboratory,
pituitaries were washed several times with fresh MEM, and the posterior
lobes were discarded. Anterior pituitaries (3–4 pooled per experiment)
were enzymatically and mechanically dissociated into single cells
following a protocol described elsewhere (30, 31). In brief, anterior
pituitary glands were cut into fragments of 1–2 mm3,
decanted, and then exposed sequentially to 0.3% trypsin (type I),
0.1% collagenase (type V), 0.1% soybean trypsin inhibitor I, 2
µg/ml DNAse, and Ca2+/Mg2+-free HBSS with
EDTA (2 mM and 1 mM), followed by a short
mechanical dissociation. Cell suspension was then filtered through a
nylon gauze (100 µm mesh), centrifuged, and suspended again in HBSS.
Cellular viability, as estimated by the trypan blue exclusion test in a
Neubauer chamber, was higher than 90%.
Cell culture
Dispersed cells were plated at a density of 300,000 cells/200
µl MEM into 24-well culture plates (for secretion experiments) or
35-mm culture dishes (for GH mRNA quantification) and allowed to attach
to the plate for 45–60 min in a humidified atmosphere containing 5%
CO2. Subsequently, cell cultures received 800 µl/well or
1,800 µl/dish MEM supplemented with FBS (10% final), as well as
antibiotic-antimycotic solution (1%), and gentamicin sulfate (50
µg/ml). After 48 h of culture, medium was replaced by fresh
MEM-FBS. Cultures were maintained for 3 days before treatments.
Secretion experiments
On the day of the experiment, cells cultured in 24-well plates
were incubated for a 4-h period with serum-free MEM and then treated
for 4 h with 10-9 M PACAP38 or PACAP27
alone or in combination with the corresponding test substance. Blockers
of the different second messengers were added to the incubation medium
90 min before PACAP treatment at the concentrations indicated in figure
legends. To test PKC activity, two types of experiments were performed:
1) short-term incubations (4 h) with increasing doses of TPA alone
(10-6–10-9 M); and 2) long-term
incubations (22 h) with 10-8 M TPA to achieve
PKC depletion before 4-h treatments with PACAP38, PACAP27, or
forskolin. After the experimental treatments, media were collected from
the culture wells, centrifuged for 5 min to remove cell debris, and
then the liquid fraction was stored at -20 C until its pGH content was
assayed by enzyme immunoassay, as previously described (31). All
treatments were carried out in quadruplicate in individual experiments,
and each experiment was repeated at least four times. GH release during
the 4-h treatment with secretagogues is expressed as a percentage of
hormone release in wells that received medium alone.
GH mRNA quantification
Cells cultured in 35-mm plastic dishes were preincubated for
4 h in serum-free MEM. Subsequently, incubation continued for
16 h in the presence of 10-9 M PACAP38 or
PACAP27 alone or in combination with the corresponding test substance.
As for the secretion experiments, blockers of the different second
messengers were added 90 min before PACAP administration. At the end of
the treatments, the medium was removed and cells were rinsed with 0.01
M PBS (pH 7.2). Thereafter, cells were fixed in the culture
dishes with 4% paraformaldehyde in PBS, dehydrated in a graded series
of ethanol and kept dry at -70 C until use. Three culture dishes were
processed per experiment for in situ hybridization, and each
experiment was repeated at least three times. GH mRNA accumulation
during the 16 h treatment with secretagogues is expressed as a
percentage of basal GH mRNA content in cells incubated with medium
alone.
The in situ hybridization procedure was performed as described previously (18). The probe used was a 26-base oligonucleotide specific to a region in the pGH mRNA encoding amino acids 102–110 of the protein, which was digoxigenin-labeled at its 3'-end using a Digoxigenin Oligonucleotide Labeling kit. In brief, cells were rehydrated, rinsed with PBS, and sequentially passed through 1% Triton X-100, 5 µg/ml proteinase K, and postfixed in 4% paraformaldehyde before addition of the hybridization mixture. Hybridization buffer [50% deionized formamide, 5 x Denhardt’s solution (1% Ficoll type I, 1% polyvinylpyrrolidone, 1% BSA in H2O), 5 x SSPE (0.75 M NaCl, 0.05 M NaH2PO4, 5 mM EDTA, pH 7.4), 4% dextran sulfate, 0.1% SDS, 250 µg/ml heat-denatured salmon sperm DNA (DNA disodium salts, type III), 200 µg/ml yeast transfer RNA, and 2 µg/ml polyriboadenosine] containing the digoxigenin-labeled probe at 35 ng/200 µl was placed in the culture dishes. After overnight hybridization in a humid chamber at 37 C, cells were sequentially rinsed with 2 x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.4), 1 x SSC, and 0.5 x SSC. Then, cells were washed in buffer I (0.1 M Tris, 0.15 M NaCl, pH 7.5), and sequentially incubated in the same buffer containing 2% BSA and 0.3% Triton X-100, and with the alkaline phosphatase-labeled antidigoxigenin F(ab) fragment diluted 1:500 in buffer I containing 1% BSA and 0.3% Triton X-100. Cell-bound alkaline phosphatase activity was visualized by incubating the cells with buffer II (0.1 M Tris, 0.1 M NaCl, 0.05 M MgCl2, pH 9.5) containing 4.5 µl/ml nitroblue tetrazolium salt, 3.5 µl/ml 5-bromo-4-chloro-3-indolyl phosphate, and 0.24 mg/ml levamisole. Finally, samples were mounted in glycerol:buffer I (1:1).
Densitometric quantification of intracellular GH mRNA content in single somatotropes was performed using an image analysis system. Specifically, a Universal microscope (Zeiss, Oberkochen, Germany) was connected by a CCTV camera (Sony, Tokyo, Japan) to a Pentium computer equipped with digitizer cards and the Software package for Image Analysis Visilog (version 4.1; Visilog, Noesis, France). Microscopic fields with hybridized pituitary cells were randomly selected. Staining intensity (optical density) of each individual cell was measured, and the relative amount of GH mRNA per somatotrope was calculated in terms of Integrated Optical Density (IOD) and expressed in arbitrary units. For each experiment, at least 45 GH mRNA-positive cells per culture dish were analyzed. An average IOD value of nonstained cells was calculated in each dish (blank value) and substracted from the IOD value of single positive cells in that dish. To avoid variations on the IOD due to factors such as probe labeling, illumination, or focusing, samples from the same experimental set were simultaneously hybridized and measured within the same session.
Statistical analysis
Statistical analyses were carried out with the program
Statistica for Windows (Statsoft, Inc., Tulsa, OK). A one-way ANOVA
followed by a post hoc Duncan’s test was applied to compare
experimental treatments. Differences were considered to be significant
at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B). The AC activator forskolin
(FK; 10 µM) induced a significant increase in GH release
that was completely blocked by coincubation in the presence of the AC
inhibitor MDL 12,330 A (10 µM) (Fig. 1A). Similarly, MDL
12,330 A totally reversed PACAP38- and PACAP27-stimulated GH release
without affecting basal release by itself (Fig. 1A). To determine
whether this AC-dependent GH release was mediated through the
subsequent activation of PKA, the specific inhibitor of this enzyme,
H89 (15 µM), was employed. As shown in Fig. 1B, H89
reduced the increases in GH elicited by FK, PACAP38, and PACAP27 to
control levels but did not modify basal release. These results indicate
that AC and PKA are actually involved in transducing the stimulus of
both PACAP38 and PACAP27 on porcine somatotropes to release GH.
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) and PACAP27-stimulated
GH release (Fig. 2B), whereas it did not alter basal GH secretion (Fig. 2, A and B). Next, we investigated the possible involvement of L-type
voltage-sensitive Ca2+ channels (VSCC) in the entry of
extracellular Ca2+ induced by both PACAPs. For this
purpose, the effects of two selective blockers of L-type VSCC were
tested. As shown in Fig. 2 (A and B), both nifedipine (1
µM) and verapamil (1 µM) specifically
abolished the GH responses to PACAP38 (Fig. 2A) and PACAP27 (Fig. 2B)
without altering basal GH release from porcine pituitary cells. Thus,
both PACAP38- and PACAP27-induced GH release depends on extracellular
Ca2+ entry through L-type VSCC.
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). In
contrast, it partially inhibited the increase in GH evoked by PACAP38
(Fig. 2C), thereby suggesting that intracellular Ca2+
participates in the response of somatotropes to this peptide.
Role of PLC/PKC pathway in PACAP38- and PACAP27-stimulated GH
release
To examine whether PLC mediates PACAP-induced GH release, a
specific inhibitor of this enzyme, U73122 (5 µM), was
used. Treatment with U73122 did not affect basal or PACAP27-stimulated
GH release (Fig. 3). However, it
partially diminished PACAP38-induced GH secretion (Fig. 3).
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, TPA increased GH release in a dose-dependent manner, thereby indicating
that PKC is able to stimulate somatotrope secretory activity. Secondly,
long-term exposure to TPA was performed to cause PKC depletion (Fig. 4B). Such a depletion was confirmed because pretreatment with
10-8 M TPA for 22 h prevented the
response of somatotropes to a subsequent 4-h exposure to the same dose
of TPA. However, PACAP38 and PACAP27 exerted similar stimulations in
TPA- and MEM-preincubated cells, thus suggesting that the effects of
both PACAPs on GH release were not dependent on PKC activity (Fig. 4B).
Finally, PKC depletion did not modify FK-stimulated GH secretion.
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). Similarly, incubation
with the L-type-channel antagonist verapamil (1 µM)
reduced the accumulation of GH transcripts induced by PACAP38 and
PACAP27 to control levels (Fig. 5). In contrast, none of the inhibitors
used modified basal levels of GH mRNA when administered alone. These
results suggest that AC/cAMP/PKA and extracellular Ca2+ are
required by both PACAPs to increase GH transcription.
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). On the other hand, inhibition of PKC
by phloretin (Ph; 20 µM) did not significantly reduce
PACAP38- and PACAP27-induced GH mRNA increases, although such increased
mRNA values were numerically, but not significantly higher than
transcript levels observed in cells treated with Ph alone (Fig. 6).
Specific inhibition of PKC activity by Ph was confirmed by its ability
to block TPA-stimulated GH release (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well established that cAMP production is a key step in the cascade of intracellular signals leading to GH release and gene expression in somatotropes (32, 33). Here, we found that both dbcAMP and the AC activator forskolin stimulated GH release from porcine somatotropes. These results confirm and extend previous observations that showed the analog 8-Br-cAMP to stimulate GH release in cultures of piglet pituitary cells (34). Moreover, by using the specific AC inhibitor MDL 12,330A we have demonstrated that AC activation and the subsequent cAMP production is a necessary step for both PACAP38 and PACAP27 to elicit GH release because this agent completely abolished their stimulatory effects. Likewise, we found that PKA inhibition by H89 completely blocked PACAPs- and forskolin-induced GH release in a similar manner, indicating that the next step in this transduction pathway, i.e. cAMP-mediated activation of PKA, is also required by both PACAPs to stimulate GH secretion. In rat somatotropes, indirect evidence from intracellular Ca2+ measurements in isolated somatotropes has led to the concept that the AC/cAMP/PKA pathway mediates GH-releasing ability of PACAP38 because the presence of the cAMP antagonist and PKA blocker RpcAMPs prevented the peptide from inducing increases in [Ca2+]i (27, 28). Similarly, blockade of PKA with H89 greatly diminished PACAP38- and PACAP27-induced [Ca2+]i increases in porcine somatotropes (29). Consistent with these findings, our present results demonstrate that both PACAP38 and PACAP27 stimulate GH release by porcine somatotropes through the required activation of AC and PKA.
Ca2+ plays an essential role as second messenger in somatotropes in response to a number of stimuli including PACAP (2, 26, 27, 28, 32, 33, 35). Hence, we evaluated the possible role of extra- and intracellular Ca2+ in the secretory response of porcine somatotropes to PACAP38 and PACAP27. Our results revealed that extracellular Ca2+ entry is a requisite step for the stimulatory action of both peptides on GH release. Furthermore, by using the specific channel antagonists nifedipine and verapamil, we demonstrated that extracellular Ca2+ entry occurs through L-type VSCC in both cases. Earlier reports had shown the ability of PACAP38 to increase free cytosolic Ca2+ in frog (36) and rat (26, 27, 28, 37) somatotropes, and in this latter species, this effect was found to be extracellular Ca2+- and PKA-dependent (26, 27, 28, 37). These two signals are likely interrelated because phosphorylation of L-type VSCC by PKA is known to be required for channel opening in response to depolarization in somatolactotropic cells (38). In line with these observations, we have recently reported that the ability of PACAP38 and PACAP27 to increase [Ca2+]i in pig somatotropes is greatly impaired by extracellular Ca2+ removal, by addition of verapamil, and by inactivation of PKA (29). Thus, in view of these and our present findings, it seems reasonable to suggest that PACAPs-stimulated GH release from porcine somatotropes is mediated by the sequential activation of the AC/cAMP/PKA system which, in turn, would trigger extracellular Ca2+ entry through L-type VSCC by channel phosphorylation. Nevertheless, it must be noted that the action of PKA in response to PACAP may not be restricted to VSCC. As it has been recently shown in GH3 cells, PKA phosphorylation of voltage-gated Na+ channels may also be required to facilitate Na+ entry and the subsequent depolarization which, in turn, would activate extracellular Ca2+ entry through L-type VSCC (39).
Interestingly, results obtained using thapsigargin strongly suggested that mobilization of Ca2+ from intracellular stores contributes partially, albeit significantly, to PACAP38-, but not PACAP27-induced GH release from porcine somatotropes. This finding was somewhat unexpected for two reasons. First, current evidence suggests that PACAP38 action on normal and tumoral rat somatotropes was not linked to intracellular Ca2+ (2, 28). Second, because extracellular Ca2+ deprivation completely blocked the secretory response of porcine somatotropes to PACAP38, we did not expect also intracellular Ca2+ to be required. Notwithstanding, results on the participation of the IP pathway supported and extended this observation. Indeed, selective blocking of PLC caused a partial inhibition of GH release induced by PACAP38, without affecting basal GH secretion or significantly reducing PACAP27 stimulatory effect. It is worth noting the striking parallelism between the present results and our recent findings that intracellular Ca2+ and PLC partially contribute to PACAP38-, but not to PACAP27-induced [Ca2+]i increases in single porcine somatotropes (29). Thus, taking these and our present results together, it can be suggested that PACAP38-stimulated GH release is partially dependent on PLC activation and subsequent mobilization of internal Ca2+ stores, whereas these mechanisms would not mediate the effect of PACAP27. On the contrary, although we found that PKC activation by TPA leads to GH release in porcine somatotropes, our results on PKC-depleted cells indicate that this enzyme does not participate in the GH release induced by PACAP38 or PACAP27 in this cell type, similar to that described in rat somatotropes (12).
Analysis of the intracellular mechanisms that mediate the effects of PACAP38 and PACAP27 on the levels of GH mRNA in individual porcine somatotropes was performed by means of nonradioactive in situ hybridization. Similar to that observed for GH release, we found that selective inhibition of AC or PKA resulted in suppression of PACAPs-induced GH mRNA increases. These results indicate that the stimulatory effects of both PACAPs on GH mRNA accumulation are completely dependent on AC and PKA activities. Likewise, we observed that extracellular Ca2+ entry through L-type VSCC was also a necessary step for such stimulatory action. Conversely, PLC blockade did not affect the stimulatory capacity of PACAP27, although it diminished partially the GH mRNA increases induced by PACAP38. In addition, results on PKC inhibition are not conclusive, but a possible participation of PKC in this effect should not be discarded. Our present findings are the first to describe the possible mechanisms mediating the effects of PACAP on GH gene expression. In cultures of rat pituitary cells, PACAP38 has been shown to increase GH mRNA levels (13, 41), yet the mechanisms involved in those effects were not addressed. Soto et al. (42) have reported that PACAP38 increases mRNA levels of the transcription factor Pit-1/GHF-1, which is known to control GH gene expression (43). This suggests a possible mechanism by which PACAP could regulate GH transcription. In addition, the group of Bancroft (44) has described that PACAP increases PRL gene expression in the clonal cell line GH3 through a PKA-dependent mechanism that involves a protein related to CREB [cAMP responsive element (CRE)-binding protein], a factor that also regulates GH gene expression (43). According to the present results, the mechanisms that mediate PACAPs-induced increases of porcine GH mRNA levels are dependent predominantly, but not exclusively, on the activation of AC/cAMP/PKA system and extracellular Ca2+ entry, because PLC is partially required by PACAP38 to exert its full effect. Therefore, it can be suggested that multiple intracellular signaling pathways interact to control porcine GH mRNA levels in response to PACAP.
The possibility that PACAP38 can simultaneously activate cAMP- and IP-dependent signaling pathways in the same pituitary cell type is not novel but has been observed previously in mouse melanotropes (45) and in clonal rat gonadotropes (2, 28, 46, 47). One possible mechanism that may contribute to such dual activation relates to the diversity of PVRs. Of the three major PVRs described to date, both PACAPs act through VPAC1 and VPAC2 types to activate prevalently AC (1, 2, 28). Conversely, various splice variants of the rat PAC1 type (PAC1 short and PAC1hop subtypes) activate both AC and PLC upon PACAP38 binding, whereas similar PACAP27 doses only activate the AC pathway, and micromolar concentrations of this peptide are required to enhance PLC activity (22). Initial studies suggested that the response of somatotropes to PACAP was mediated by VPAC2 or VPAC1, but not PAC1 (2, 23, 27, 28). However, recent evidence indicates that the splice variants PAC1s and PAC1hop are indeed expressed in normal and clonal rat somatotropes (24, 44, 48). Although the type of PVRs expressed in porcine pituitary has not yet been determined, a type I PACAP-binding site, analogous to the rat PAC1, has been isolated from pig brain (49, 50). According to our results that both PACAP38 and PACAP27 require activation of AC, but that only the former requires PLC activation in porcine somatotropes, it is tempting to speculate that the type(s) of PVR(s) expressed by this cell type would share some functional characteristics with those found for rat PAC1s and/or PAC1hop. Likewise, the putative PVR of porcine somatotropes should differ from human PAC1 subtypes described recently, because PACAP27 and PACAP38 similarly activate AC and PLC through these receptors (51, 52). Nevertheless, we must introduce the caveat that small, nonsignificant reductions in PACAP27-induced GH release and GH mRNA accumulation were observed in the presence of PLC and intracellular Ca2+ inhibitors. Thus, the possibility that PACAP27 could also activate the PLC/intracellular Ca2+ pathway in porcine somatotropes under other conditions (e.g. higher PACAP27 doses, physiological status of the pituitary donor) should not be definitely discarded.
In a previous study (18), we observed that the stimulatory action of PACAP38 and PACAP27 on GH release and mRNA levels displayed different dose- and time-related patterns, respectively. Thus, PACAP27 stimulated GH release in a dose-dependent manner, whereas PACAP38 stimulation did not follow such a pattern. Likewise, PACAP27-induced GH mRNA increased more rapidly (8 h) than PACAP38 (16 h). Our present observations on the intracellular mechanisms employed by each peptide to increase GH release and mRNA levels in somatotropes might contribute to explain these differential actions. As it has been suggested in clonal gonadotropes, concurrent activation of cAMP and IP signaling systems by PACAP in the same cell can lead to cross-talk among individual components of each pathway (47). That cross-talk processes occur in porcine somatotropes is supported by our observation that contribution of PLC and intracellular Ca2+ to the effects of PACAP38 in GH release and GH mRNA levels were dependent on extracellular Ca2+ entry. In fact, an analogous mechanism operates for PACAP38-induced increases in [Ca2+]i in this cell type (29). Thus, it is conceivable that activation of cAMP- and IP-dependent pathways in porcine somatotropes by PACAP38 could result in cross-talk processes that may account for the differences with respect to PACAP27. Porcine somatotropes could thus provide a useful, nontumorous cell model to investigate the mechanisms of differential activation of intracellular signals by PACAP38 and PACAP27, and of how these signals (co)operate to control GH release and gene expression.
In summary, our results indicate that both PACAPs stimulate GH biosynthesis and release in porcine somatotropes mainly through activation of the AC/cAMP/PKA pathway, which is positively coupled to extracellular Ca2+ influx through L-type VSCC. In addition, PLC and Ca2+ from intracellular stores contribute -yet only partially- to PACAP38-stimulated GH release and transcription. Our findings demonstrate, for the first time, the existence of differences in the second messengers required by PACAP38 and PACAP27 to stimulate normal, nontumorous somatotropes. Moreover, these differences could explain the previously described dose- and time-dependent divergences in the actions of both peptides on porcine somatotropes. Existence of such differential actions is likely to be related to the type(s) of PACAP-receptor subtype(s) expressed in porcine somatotropes and in their affinity for PACAP38 and PACAP27, G protein coupling, and/or ability to activate diverse signal transduction pathways.
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Acknowledgments |
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Footnotes |
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Received April 1, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells. Endocrinology 134:2599–2605[Abstract]
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cells. Ann NY Acad Sci 805:112–121[Abstract]
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