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Endocrinology Vol. 139, No. 11 4590-4597
Copyright © 1998 by The Endocrine Society

ARTICLES

Protein Phosphatase 2A Plays a Role in the Suckling-Induced Changes in the Responsiveness of Pituitary Mammotropes1

Andrea Murányi, Pál Gergely, Márton I. K. Fekete and György M. Nagy

Department of Medical Chemistry, University Medical School of Debrecen (A.M., P.G.), Debrecen; and EGIS Pharmaceuticals Ltd. (M.I.K.F.) and the Neuroendocrine Research Laboratory, Department of Human Morphology and Developmental Biology, Semmelweis University Medical School (G.M.N.), Budapest, Hungary H-1094

Address all correspondence and requests for reprints to: Dr. György M. Nagy, Neuroendocrine Research Laboratory, Department of Human Morphology and Developmental Biology, Semmelweis University of Medicine, Budapest, Tüzoltó u.58, Hungary H-1094. E-mail: nagy-gm{at}ana2.sote.hu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is well established that PRL secretion is under a tonic inhibition exercised by the hypothalamic dopamine (DA). One feature of this regulation is an immediate withdrawal reaction (elevation of PRL release) of mammotropes after disruption of hypothalamic influence. Although plasma PRL rises rapidly, the suckling stimulus does not cause an acute diminution of hypothalamic DA, but, as we have previously demonstrated, it results in an almost immediate (within 10 min) desensitization of mammotropes as indicated by the change in dose response of DA to inhibit PRL release. Our present investigations relate to the phenomenon of this change in responsiveness of PRL cells. This was accomplished by using the reverse hemolytic plaque assay to evaluate the secretory characteristics of individual PRL secretors derived from lactating rats either before or after a 10-min suckling stimulus. To investigate the mechanism of these changes, the binding characteristics of [3H]spiperone on pituitary membranes from nonsuckled and suckled rats have been compared, and the possible involvement of dephosphorylating enzymes was tested by using okadaic acid (OA) in a dose of 2 nM that preferentially and selectively inhibits protein phosphatase-2A (PP2A) activity. We have also determined the activities of PP1 and PP2A in pituitary tissue samples as well as in enzymatically dispersed cells. Mammotropes from nonsuckled rats exhibited a depression of PRL release after both DA and OA treatment and an elevation after withdrawal of DA. This suggests that the secretory response of mammotropes obtained from nonsuckled rats still shows those two responses that are characteristic of the tonic inhibitory regulation. In contrast, superimposition of suckling in vivo or application of OA together with DA pretreatment in cells from nonsuckled rats in vitro resulted in a disappearance of the dissociation-induced elevation of PRL release, indicating an abolishment of the tonic inhibitory action of DA. Evidence is also presented that the PP2A, but not the PP1, activity of the anterior lobe is significantly lower after a 10-min suckling stimulus. Moreover, DA is able to decrease PP2A activity in dispersed pituitary cells obtained from nonsuckled, but not from suckled, animals. In contrast, there were no differences in either the affinity or the number of binding sites between nonsuckled and suckled rats. Taken together, our results suggest that the suckling-induced decrease in PP2A activity plays a role in the uncoupling of D2 receptors on mammotropes from the tonic inhibitory signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL documented that the suckling stimulus primes or sensitizes the anterior lobe of the pituitary gland to PRL-releasing stimuli such as TRH or crude hypothalamic extracts. This priming phenomenon was first detected in vivo (1, 2, 3). Moreover, these early studies have shown that a DAergic mechanism regulates the first (approximately 10–15 min after suckling has been initiated), but not the second, phase of PRL secretion during a prolonged (30–60 min) suckling stimulus (1). It has been suggested that the rapid and concomitant development of these changes in pituitary responsiveness are not independent (1, 2, 3).

We have recently demonstrated that the suckling-induced changes in responsiveness of pituitary tissue are manifest at the cellular level of mammotropes. Dopamine (DA) was only marginally effective at inhibiting (4, 5), but TRH and angiotensin II were far more effective at stimulating (5, 6), PRL release from short term cultured pituitary cells derived from acutely suckled mothers compared with cells obtained from mothers separated from their pups for 4 h. Population analysis revealed that there is a proportional shift away from those cells most susceptible to inhibition by DA (desensitization) and toward those cells most responsive to stimulatory secretagogues (sensitization) due to a 10-min suckling stimulus (5, 6).

Long term desensitization/sensitization processes by which many cells adapt to prolonged activation or inactivation of receptor-mediated signal transduction mechanisms generally involve changes in the receptor numbers (up- or down-regulation) and/or in the gene expression of molecules in the signal transduction cascade (7). In contrast, the suckling-induced desensitization/sensitization of mammotropes are extremely rapid and, therefore, most likely involve alterations in protein phosphorylation. Virtually all types of extracellular signals are known to produce their physiological effects by regulating the state of phosphorylation of specific phosphoproteins in their target cells (8, 9). A change in the phosphorylation of a protein can be achieved through increases or decreases in the activity of phosphorylating protein kinases (PKs) and/or dephosphorylating protein phosphatases (PPs), respectively. Although the role of the PKs in the secretory function of the pituitary gland has been widely explored (10), the role of the PPs has been mostly ignored. Multiple forms of phosphoserine/phosphothreonine-specific PPs have been recognized in mammalian tissues (11, 12, 13). They can be categorized into the major classes PP1, PP2A, PP2B, and PP2C according to their substrate specificity and their sensitivity to inhibitory proteins (11, 12). For example, PP1 is inhibited by the thermostable proteins, termed inhibitor-1 and inhibitor-2, whereas PP2A is insensitive to inhibitor-1 and -2. The activity of PP2A in tissue extracts is completely inhibited by 1–2 nM okadaic acid (OA), whereas complete inhibition of PP1 requires 1 µM OA (14).

To investigate the possible involvement of dephosphorylating enzymes in the suckling-induced changes in DA responsiveness, PRL release has been studied in the presence of OA in cells obtained from lactating rats either separated from their pups for 4 h (nonsuckled) or when 4-h separation was followed by a 10-min suckling period (suckled). The role of PP2A in the elevation of PRL release in response to interruption of DA inhibition (withdrawal response) has also been tested in cells from both nonsuckled and suckled mothers. To investigate a possible direct involvement of dephosphorylating enzymes in the desensitization/sensitization cascade, we determined the activity of two PPs, PP1 and PP2A, in pituitary tissue samples as well as in dispersed cells with and without DA treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Primiparous lactating rats (bred in our animal facilities from Sprague-Dawley stock originally obtained from Hanover, Germany) with standardized litter size (eight pups from the first day postpartum) were used on days 6–10 of lactation. The animals were housed in an air-conditioned room at 21–23 C in individual cages with alternating 14 h of light and 10 h of darkness. They received rat pellets and water ad libitum. On the day of the experiment, pups were removed from their mothers at 0900 h. Four hours later (at 1300 h) some mothers were reunited with their pups for 10 min (suckled group), whereas the others (nonsuckled group) were not. Both groups of animals were killed by decapitation within 5 sec after being taken from their cages.

Materials
Phosphorylase b was purified from rabbit skeletal muscle (15) and converted to 32P-labeled phosphorylase a (~0.9 mol phosphate/mol subunit) according to the method of Cohen et al. (16). 32P-Labeled myosin light chain (~1 mol phosphate/mol subunit) was prepared as described by Erdödi et al. (17). Inhibitor-2 was purified from rabbit skeletal muscle (18). Dithiothreitol, bacitracin, EGTA, phenylmethylsulfonylfluoride (PMSF), and soybean trypsin inhibitor were purchased from Sigma Chemical Co., Inc. (St. Louis, MO). Benzamidine was from Merck (Rahway, NJ), and OA was obtained from Life Technologies (Grand Island, NY). ({gamma}-32P]ATP was purchased from the Hungarian Isotope Institute (Izinta, Hungary) or ICN Biomedicals, Inc. (Irvine, CA). [3H]Spiperone was obtained from Amersham International (Aylesbury, UK). Other chemicals used in these studies were of the highest grade available commercially.

Receptor binding assay
After separating the neurointermediate lobe from the anterior lobe (AL), the later were homogenized in 2 ml ice-cold 0.05 M sodium phosphate buffer (pH 7.4) in a glass-Teflon tissue homogenizer and suspended in 2 ml phosphate buffer containing 100 µM PMSF, 1 mM dithiothreitol, 50 µg/ml soybean trypsin inhibitor, and 40 kallikrein inhibitor units/ml aprotinin (Gordox, Richter Gedeon Rt., Budapest, Hungary). The affinity and binding capacity of DA receptor type D2 in the AL homogenate were determined using [3H]spiperone receptor binding assay. Freshly prepared homogenates (protein concentration, 2.0–2.5 mg/ml) were incubated with 7–12 concentrations of [3H]spiperone (0.5–25.6 nM; SA, 20 Ci/mmol; Amersham International) in a final volume of 200 ml in half of the samples at the presence of haloperidol (10-6 M) to determine nonspecific binding. Incubations were started by adding the homogenate, then were continued for 30 min at 37 C to achieve the steady state. Bound radioactivity was separated from free on a Skatron cell harvester (Sterling, VA) through Whatman GF/B filters (Clifton, NJ) with ice-cold sodium phosphate buffer (pH 7.4). Filters were put into 2 ml Ultima Gold (Packard, Downers Grove, IL) scintillation cocktail. The results were evaluated using Prism 2.00 software (GraphPad Software, Inc., San Diego, CA).

Pituitary cell dispersion and reverse hemolytic plaque assay (RHPA)
The pituitary glands from both groups of lactating rats were removed under aseptic conditions. The anterior pituitaries were separated from neurointermediate lobes. Tissue fragments of anterior lobes were dispersed with trypsin. RHPA was used for detecting hormone secretion of individual cells. This assay was conducted as described in detail previously (3, 4, 5). Immediately after dispersion, cells were washed several times with DMEM containing 0.1% BSA, penicillin G (100 U/ml), and streptomycin sulfate (100 µg/ml). The monodispersed cells (final cell number, 3 x 104 cells/ml) were mixed with protein A-coupled sheep red blood cells, and approximately 30-µl aliquots were infused into Cunningham chambers to form a cell monolayer during a 45-min preincubation period (at 37 C in a humid, 5% CO2 and 95% air atmosphere). Next, chambers were washed with DMEM-0.1% BSA to remove unattached cells and were filled with the same medium containing anti-PRL antiserum at final dilution of 1:50 with or without 1 µM DA. Subgroups of pituitary cells were preincubated with or without 2 nM OA for 30 min [because OA at this concentration specifically inhibits PP2A activity (11)] before initiation of the plaque assay. Incubation of cells with PRL antiserum was conducted for 1 h, followed by 30-min treatment with guinea pig complement (at a final dilution of 1:50). The reaction was terminated by infusion of 2% glutaraldehyde in physiological saline. The following parameters of PRL secretion were measured: 1) number of plaques [percentage of plaque-forming cells (PFC)] = number of secretory mammotropes, 2) mean plaque area (MPA; square microns) = mean PRL secreted/mammotropes, and 3) total secretion index (square microns) = MPA x PFC. Two hundred cells or plaques on duplicate slides in each experimental group were measured.

Preparation of tissue or cell extracts
Pituitary glands were removed from the rats and for immediate tissue measurements were suspended in 40 mM Tris-HCl (pH 7.4) buffer at 0–4 C. Enzymatically dispersed pituitary cells (5 x 105 in each experimental group) were incubated for 1 h with or without 10-6 M DA, centrifuged, then suspended in the same buffer. For the assay of PP1 and PP2A, tissue samples or cells were sonicated in 0.6 ml buffer containing 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 0.5% 2-mercaptoethanol, 1 mM PMSF, 2 mM benzamidine, and 1 mM o-phenantroline 10 times for 2 sec each time with a Branson sonifier (Branson, Danbury, CT; model 250) at 0–4 C. The sonicate was centrifuged at 12,000 x g for 5 min, and the clear supernatant was used for enzyme assay. Protein contents were determined as described by Bradford (19) using BSA as a standard.

Assay of protein phosphatases
The assay mixtures contained 50 mM Tris-HCl (pH 7.4), 0.1 mM EGTA, 0.1% 2-mercaptoethanol (buffer A), tissue or cell lysate, and 10 µM [32P]phosphorylase a with 5 mM caffeine or 5 µM [32P]myosin light chain as phosphosubstrate. The assay was performed according to the method of Erdödi et al. (17) with or without 1 µM inhibitor-2 and 2 nM OA. The amount of phosphatase (in dilution of samples) was chosen in the assays such that no more than 30% of the substrate would be converted during the incubation time. One unit of activity is the amount of the enzyme that catalyzes the dephosphorylation of 1.0 µmol phosphorylase a/min.

Statistical analysis
Results are expressed as the mean ± SEM. Statistical differences were calculated with Student’s t test for independent random samples or using ANOVA and Dunnett’s multiple range post test, as appropriate. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of the length of the incubation time during the RHPA on the secretory functions of mammotropes
Anterior pituitary cells of nonsuckled and suckled lactating rats were subjected to RHPA immediately after cell dispersion. This assay permits microscopic demonstration of both the number of secretory mammotropes (percentage of PRL cells) and the amount of PRL released by individual cells (MPA) as area of hemolysis (plaques) surrounding pituitary cells (1, 2). Compared with cells obtained from mothers suckled for 10 min (Table 1Go), both the number of PFC and the MPA that developed under basal conditions were significantly higher in nonsuckled animals when the assay was run for a shorter incubation period (30–60 min). Using a longer incubation time (90–120 min), these differences in basal secretion disappeared; however, the appearance of two subpopulations reflecting two secretory modes of mammotropes clearly demonstrated a functional difference between nonsuckled and suckled rats.


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  		Table 1. Effect of the length of the incubation time on the percentage of plaque-forming PRL cells (PFC) and the mean plaque are (MPA) of mammotropes from nonsuckled and suckled mothers

 
Effect of DA or OA on PRL release of mammotropes
Dispersed pituitary cells from both nonsuckled and suckled dams were treated with 1 µM DA or 2 nM OA, and PRL release was measured by RHPA. As shown in Fig. 1Go, a brief nursing episode completely prevented the degree to which a maximal dose of DA [1 µM, established in several previous experiments (4, 5)] inhibited PRL plaque sizes (P < 0.05). OA (2 nM) reduced PRL release (P < 0.05), similar to the effect of DA on cells derived from mothers not allowed to be suckled. When OA and DA were tested together, there was no further decrease in the amount of PRL released (Table 2Go). A short exposure to suckling also prevented the reducing effect of OA on the average size of plaques that formed around individual PRL cells (Fig. 1Go). Practically the same results could be detected using another, less selective PP inhibitor caliculin A (data not shown).



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  		Figure 1. Effect of DA (1 µM) or OA (2 nM) on PRL release of pituitary mammotropes obtained from nonsuckled or suckled lactating rats. Subgroups of pituitary cells from nonsuckled and suckled dams were preincubated with and without 2 nM OA for 30 min before initiation of the RHPA by infusing solutions containing antibody and appropriate treatments (1 µM DA or 2 nM OA). Each column represents the mean (±SE) of four separate experiments. The mean area of PRL plaques under basal conditions were 16,825 ± 3,107 and 10,320 ± 3,167 µm2 for nonsuckled and suckled groups, respectively. Asterisks indicate a significant difference (P < 0.05) vs. untreated controls.

 

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  		Table 2. Effects of DA and OA alone or in combination on invitro PRL release from pituitary mammotropes

 
Effect of OA on DA removal-induced PRL release
Interruption of DA (1 µM) treatment after 2 h induced a significant elevation in the mean amount of PRL (MPA) released by mammotropes obtained from nonsuckled mothers but not in those from suckled animals (Fig 2AGo). Treatment of cells from a nonsuckled rat with OA (2 nM) during the 2-h preincubation period did not result in a significant reduction of the MPA. The presence of OA during the preincubation period completely prevented the DA withdrawal-induced increase in PRL secretion of mammotropes (Fig 2BGo).



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  		Figure 2. Effect of the removal of DA from the culture medium on cells obtained from both nonsuckled (control value, 14,896 ± 2,291 µm2) and suckled (control value, 11,555 ± 1,891 µm2) mothers (A) and the effect of OA on the DA removal-induced withdrawal signal detected in cells obtained from nonsuckled rats (control value, 24,472 ± 3,291 µm2; B). Two hours of preincubation with medium, DA (1 µM), or OA (2 nM) alone or in combination was followed by 2-h RHPA without any secretagogues. Each column represents the mean (±SE) of three independent experiments. Asterisks indicate a significant difference (P < 0.05) vs. untreated controls.

 
Effect of suckling stimulus and DA on PP1 and PP2A activity of dispersed pituitary cells
PP1 and PP2A activities in anterior lobes obtained from nonsuckled and suckled lactating rats were measured using two different substrates (phosphorylase a and myosin light chain) and gave identical results (Table 1Go and Fig. 3Go). The PP2A, but not PP1, activity of AL lysates was suppressed significantly by a 10-min suckling stimulus (Table 3Go). This change in the OA-sensitive PP2A activity could still be detected when AL cells were enzymatically dispersed and subjected to a 1-h incubation period, somewhat similar to the circumstances during the RHPA (Fig. 3Go). We next determined whether DA could affect the activities of these two enzymes in vitro in cells obtained from nonsuckled or suckled mothers. Figure 3Go illustrates that 1 µM DA decreased the PP2A activity in dispersed AL cells obtained from nonsuckled, but not suckled, mothers.



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  		Figure 3. Effect of suckling stimulus on the PP1 and PP2A activities in pituitary cells detected after dispersion and an additional 1-h incubation with or without DA (1 µM). Enzymatically dispersed pituitary cells (5 x 105 in each experimental group) were incubated for 1 h with or without DA (10-6 M), then homogenized and suspended in 40 mM Tris-HCl (pH 7.4) buffer at 0–4 C. For measurement of the PP2A activity, two different substrates (32P-labeled phosphorylase a and 32P-labeled myosine light chain) were used. The mean ± SEM of four independent (different batches of pituitary cells) measurements of enzymes activity is shown. The asterisk indicates a statistically significant difference (P < 0.05) between nonsuckled and suckled groups exposed to none or the same secretagogue. The plus sign denotes a difference (P < 0.05) between control and DA-treated groups. No significant differences were found in PP1 (inhibitor-2-sensitive) activity with these phosphosubstrates.

 

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  		Table 3. Effect of suckling stimulus on the PP1 and PP2A activities of pituitary gland compared to nonsuckled lactating rats

 
Effect of suckling on the receptor binding of [3H]spiperone
To test the effect of suckling on the D2-receptor function of the AL, a [3H]spiperone receptor binding assay was performed on membranes of ALs obtained from both nonsuckled and suckled rats. As shown in Fig. 4Go, there was no significant difference in the number of binding sites (binding capacity, 0.036 pmol in nonsuckled vs. 0.043 pmol in suckled rats) and no alteration of the affinity of this ligand (Ka, 2079 ± 492 fmol/mg protein in nonsuckled vs. 2407 ± 606 fmol/mg protein in suckled rats). This suggests that the changes in responsiveness of PRL cells do not affect ligand receptor binding and do not depend on the number of functional receptors. Rather, binding occurs more distally at the level of the receptor-signaling mechanism.



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  		Figure 4. Saturation binding curve of [3H]spiperone on pituitary glands from nonsuckled (NS) and suckled (S) lactating rats. The inset is the corresponding Scatchard plot. Representative data are shown from an experiment replicated three times with duplicate parallels each time.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first evidence presented that describes a possible mechanism responsible for the suckling-induced changes in mammotrope responsiveness. Our results indicate that a brief suckling stimulus changes the balance of the intracellular phosphorylation in mammotropes. These data clearly suggest a dynamic role for the dephosphorylating side of this balance, namely a pivotal role of PP2A in the suckling-induced abolishment of the tonic inhibitory control by DA. This intimate relationship is evidenced by the fact that the PP2A, but not PP1, activity of the AL is significantly lower after a 10-min suckling stimulus than that in nonsuckled controls. Furthermore, this striking difference in the enzyme activity due to suckling can also be detected in dispersed pituitary cells, hours after removing the glands from the animals. Moreover, parallel to its inhibitory influence on PRL release, DA is able to decrease PP2A activity in dispersed pituitary cells obtained from a nonsuckled mother, but not in that from a suckled animal. The presumption that PP2A is a key component of the D2-receptor-mediated signal transduction pathway in mammotropes is supported by our observations that OA (using a dose of 2 nM that preferentially inhibits PP2A activity), similar to the effect of DA, reduces PRL release only from cells obtained from nonsuckled rats. Furthermore, treatment of cells with OA and DA together does not cause additive inhibition of PRL secretion.

A significant increase in the release of PRL after 2-h DA pretreatment of cultured pituitary cells followed by a rapid withdrawal is another well known secretory response of mammotropes due to the D2-receptor-mediated tonic inhibitory regulation of PRL secretion (10). It can only be detected in cells obtained from nonsuckled mothers. Interestingly, a 10-min suckling stimulus as well as the presence of OA (2 nM) during the preincubation period with DA result in the complete loss of DA withdrawal-induced release of PRL. These data clearly show that desensitization of mammotropes (indicated by the reduction in the inhibitory response to DA) develops with a concurrent disappearance of the DA-mediated tonic inhibition of these cells (indicated by the lack of withdrawal signal-induced increase in PRL release). As the change in PP2A activity as well as that in DA responsiveness of PRL cells are parallel, and the large majority of D2-receptors affected by DA can be found on mammotropes only (20), our data indicate that the observed change in PP2A activity arises primarily from PRL cells. Although paracrine interactions cannot be entirely excluded, it is highly unlikely that these interactions play any role in this effect.

It is well documented that PRL secretion is constantly and severely restrained by the hypothalamus in vivo (21). Furthermore, sufficient evidence is available to support the strong conclusion that DA of hypothalamic origin, which is delivered to the adenohypophysis by way of the long and also the short portal vessels, exerts a tonic inhibition on pituitary mammotropes (21). However, investigation of a more dynamic and precise relationship of DA to pituitary PRL secretion is still a relevant issue. This is based on the fact that an inverse relationship does not exist between hypothalamic secretion of DA and pituitary secretion of PRL in most of the physiological circumstances where it is expected. For example, DA levels in hypophysial stalk plasma are 5–7 times lower in males than in females (22, 23, 24), but the plasma levels of PRL are not much different. From our point of view it is even more important that the lack of a mirror-image relationship between DA concentrations and plasma PRL also has been demonstrated in lactating rats during the suckling stimulus (25, 26, 27). The fact that a suckling stimulus can rapidly and profoundly change the responsiveness of mammotropes to PRL-inhibiting and releasing stimuli (2, 3, 5, 6, 21) without affecting either the affinity or the number of DA-binding sites highlights a possible significant and physiological role of the immediate abolishment of the tonic inhibition at the level of the signaling cascade of D2-receptors (5, 6, 28, 29). In this process, the secretory function of PRL cells may be dominantly controlled by a sequence of signal transduction mechanisms that can initiate interacting cascades of receptorial and intracellular events (14), including reduction in a dephosphorylating enzyme, PP2A. Consonant with our data, Barros et al. (30, 31) have recently shown that OA pretreatment enhances (30) and PP2A reverses (31) the delayed effect of TRH on GH3 rat anterior pituitary cells excitability, also suggesting a role for PP2A in the regulation of TRH responsiveness. Similar to these data, we have previously observed (5, 6) in cells obtained from lactating rats that a brief suckling stimulus increases mammotrope responsiveness to TRH and angiotensin II (5, 6). However, in our present study we have not investigated the role of PP2A in the change in TRH responsiveness, but all of these data together indicate that a reduction of PP2A in mammotropes may play a pivotal role in the rapid changes in responsiveness to both the inhibitory and stimulatory secretagogues.

The conspicuous difference detected in the mean area of plaques that develops under basal conditions between nonsuckled and suckled groups is not a new observation. In our previous studies (5, 6) when a 120-min incubation time has been used, and a difference in the basal secretion of mammotropes could not be detected, the frequency distributions of plaque sizes for dams not allowed to nurse before death had a bimodal distribution. There was a subpopulation of PRL cells that formed smaller plaques (released less hormone), and the other produced larger plaques (released the most PRL). The frequency of mammotropes from suckled mothers was quite different, because those cells that formed the subpopulation of larger plaques all disappeared. It has been also demonstrated (5) that the subpopulation of mammotropes that release the most hormone basally is the most responsive to the inhibitory effect of DA; consequently, there is no DA responsiveness subpopulation of PRL cells in suckled mothers. Therefore, the subpopulation of preferentially responsive mammotropes (either to DA or suckling stimulus) is one and the same. In our present experiments using a shorter incubation time (60 min), both the association-induced inhibition (pure receptorial regulation) and the dissociation-induced stimulation (tonic regulation) of PRL release can be more easily detected on cells obtained from nonsuckled rats than on those from suckled animals. Furthermore, they are in good agreement with the results of frequency distributions obtained after a longer incubation period (120 min) in our previous studies (5). Based on all of these results, the differences in the basal secretion (after 30–60 min) as well as in the frequency distribution (after 90–120 min) between these two animal models are most likely due to the same thing, namely the dissociation signal-induced elevation of PRL release from mammotropes in nonsuckled rats.

The exact mechanism of how the change in PP2A activity can affect the responsiveness of mammotropes is another open question, primarily because the substrate of PP2A has not been determined. However, it is well known that the catalytic subunits of PP1 and PP2A are complexed to several intracellular proteins in vivo, providing various possibilities for regulation (32, 33). The signal transduction cascade in mammotropes is under tonic and dominant inhibitory control of DA (21). This influence is mediated by the inhibitory G protein (Gi) known to be coupled to D2-receptors (34, 35). Hence, the alteration of the balance between the specific PK and PP activities that control the level of phosphorylation of several components of this cascade (including C-terminal portion of the receptor or subunits of Gi) might be expected to modulate mammotrope responsiveness to inhibitory or stimulatory signals (7, 34). Our results, parallel with this line of thinking, indicate that an overall control of phosphorylation in the signaling cascade through the regulation of PP2A activity may have a pivotal role in the in vivo regulation of cellular responsiveness. Without suckling, the tonic inhibitory signal is a dominant feature of hormone secretion that is characterized by the immediate elevation of PRL release. Injecting DA receptor antagonists in vivo (21) or treatment of pituitary cells from nonsuckled mothers with DA in vitro followed by removal of DA results in an immediate elevation of plasma PRL or the mean amount of hormone released by mammotropes, respectively. Immediately after initiation of the suckling stimulus, mammotropes become less susceptible to inhibition by DA (4), with a concomitant disappearance of the tonic inhibitory control. It is also evidenced by our in vivo observations that {alpha}-methyl-p-tyrosine or domperidone only slightly augments plasma PRL in suckled rats (our unpublished observation) and by the fact that the DA withdrawal-induced release of PRL cannot be detected in cells obtained from rats suckled for 10 min. It is clear that a switch between two types of dopaminergic control can occur within a few minutes due to the suckling stimulus. One of the most attractive possibilities is that it is a result of a functional uncoupling of Gi from D2-receptors due to the phosphorylation, and therefore inactivation, of the specific {alpha}-subunit. A similar ligand-induced response as well as OA-induced desensitization process and phosphorylation of Gi{alpha} have been shown in hepatocytes (36, 37, 38) and in parotid tissue of rats (39).

An alternate interpretation of our results is that the suckling-induced decrease in pituitary PP2A activity may completely occlude the DA action. Both our previous and present data suggest that occlusion may occur, as both suckling- and DA-induced changes in responsiveness have been associated with one subpopulation of pituitary mammotropes. However, our preliminary data suggest that this is not the case. As noted in the introduction, parallel with the reduction in DA responsiveness, suckling renders mammotropes more responsive to TRH, angiotensin II (5, 6), and, according to our most recent data, a direct activation of adenylate cyclase by forskolin (Horváth, M. K., B. Radnai, E. B. Horváth, M. Tóth, I. K. Fekete, and M. G. Nagy, submitted for publication). It has also been found that an inhibitory dose of DA (10-7 M) could reduce TRH-induced stimulation of PRL release in cells obtained from suckled mothers. This suggests that DA/D2-receptor coupling and one of the signaling pathways are not completely obliterated. It seems that the tonic inhibition parallel with the withdrawal signal-induced stimulation and the inhibition of the stimulus-induced PRL secretion by DA can be dissected out. A more detailed analysis of these phenomenon is presently under investigation in our laboratory.

In summary, the results of this study provide a possible intracellular basis for the priming phenomenon that develops in suckled rats. More specifically, we have shown that the suckling-induced change in mammotrope responsiveness to DA is manifest at the level of intracellular phosphorylation. Furthermore, our data suggest that the dephosphorylating side of the balance in phosphorylation is critical. More specifically, the observed difference in the activity of PP2A may be responsible for the selective development or/and disappearance of the tonic inhibitory responsiveness at the level of mammotropes. Studies aimed at further resolving this conceptionally new and suggestive mechanism are currently underway in our laboratory.


   Acknowledgments
 
We appreciate the expert technical assistance of Mrs. Ilona Rónai and Ms. Mária Mészáros.


   Footnotes
 
1 This work was supported by the Hungarian National Research Fund (OTKA 20916; to G.M.N.) and the Soros Foundation (027/1-1602 to G.M.N.; OTKA 12840 and MKM 77 to P.G.). Part of this study was presented at the Fourth International Pituitary Congress, San Diego, CA, 1996 (Abstract B59). Back

Received February 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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