Serum modulates cyclic AMP-dependent morphological changes in cultured neurohypophysial astrocytes

Brain Research Bulletin, Vol. 39, No. 2, pp. 109-114, 1996 Copyright © 1995 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/96 $15.00 + .00

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Serum Modulates Cyclic AMP-Dependent Morphological Changes in Cultured NeurohypophysialAstrocytes KATRINA D. RAMSELL, BAI-GE ZHAO, DANIEL BAKER AND PETER COBBE-I-I"1

Department of Pharmacology & Toxicology, and The Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA [Received 12 June 1995; Revised 26 July 1995; Accepted 15 September 1995] ABSTRACT: The effects of serum on the morphological plasticity exhibited by pituicytes in explant cultures of the neurohypophysis of adult rats have been examined. Cultured pituicytes are normally nonstellate, protoplasmic, amorphous cells (< 25% are stellate with a distinct cell body and phase bright processes). After incubation (90 min) of pituicyte cultures in a HEPES buffered salt solution (HBSS) supplemented with isoproterenol or forskolin, the fraction of stellate pituicytes significantly increased. The increase in the fraction of stellate cells induced by isoproterenol was not reversed by subsequent incubation in isoproterenol-free HBSS for 90 min. In contrast, after stellation was induced in cultures by exposure to forskolin (90 min), the fraction of stellate cells was significantly reduced if these cultures were incubated in forskolin-free, serum (0.5%) supplemented HBSS for the same duration. Serum also blocked the increase in the fraction of stellate pituicytes induced by forskolin. These experiments suggest that serum components may have a significant role in controlling the plasticity of neuroglial relations in the neurohypophysis previously demonstrated in vivo.

Such remodelling of neuroglial relations has also been observed in the whole neurohypophysis in vitro: incubation of isolated neurohypophyses in hypertonic physiological medium or in isotonic medium containing a fl-adrenoreceptor agonist (but not in isotonic, agonist-free medium) induces ultrastructural changes similar to those seen in the neurohypophysis of dehydrated or lactating rats [ 10,12,19]. Morphological plasticity of pituicytes is believed to be responsible for the ultrastructural changes within the neurohypophysis in vivo and in vitro [4]. Morphological plasticity of pituicytes has been observed in primary cultures of pituicytes from adult rats in response to activation of fl-adrenoreceptors [2,6,29], to activation of adenylate cyclase and soluble guanylate cyclase [ 14,15], and to increased extracellular K ÷ concentration [29]; these stimuli induce cultured pituicytes to change from a nonstellate or protoplasmic form to a stellate form. These morphological changes are similar to those described for primary cultured astrocytes from fetal neocortex and for glioma-derived astrocytes following activation of fl-adrenoreceptors [7,13,1618]. The alteration of pituicyte morphology from the nonstellate form to the stellate form in culture is thought to be analogous to the changes of pituicyte morphology that occurs in vivo in lactating and dehydrated animals, and the pituicyte culture may therefore be used as a model to examine mechanisms of morphological plasticity. All studies of plasticity of pituicyte morphology in the whole isolated neurohypophysis or in pituicyte cultures have been performed using serum-free balanced salt solutions (or solutions containing 0.05% serum). Although studies of plasticity of the morphology of fetal- or glioma-derived astrocytes have also usually been performed using serum-free media, there have been reports that serum or serum components may affect morphology of these cells [9,20]. Specifically, cells that have been induced to become stellate by activation of fl-adrenoreceptors revert to the nonstellate form more rapidly when incubated in serum-containing medium (or medium containing one of several identified serum components) than in serum-free medium. Here we demonstrate that cultured pituicytes that have been induced to become stellate by exposure to forskolin also revert rapidly to the nonstellate form when exposed to serum-containingmedium but not serum-free medium. We also show that the stellation response

KEY WORDS: Pituicyte, Serum, Cyclic AMP, Morphological plasticity.

INTRODUCTION Within the mammalian neurohypophysis, oxytocin and vasopressin are secreted into the systemic circulation from axon terminals of the magnocellular neuroendocrine cells, which are located principally in the hypothalamic supraoptic and paraventricular nuclei. The axon terminals have an intimate but plastic relationship with astrocytic glia--the so-called pituicytes-within the neurohypophysis (for review see [4,5,21]). Normally pituicyte cytoplasm is found interposed between the axon terminals and between axon terminals and the basal lamina surrounding the capillary vessels so that terminals may be completely engulfed by pituicytes. However, during prolonged elevated hormone secretion, as in dehydration or lactation, pituicytes appear to withdraw from these sites so that there is an increased amount of axon terminal membrane in direct contact with the basal lamina [22-26]. ' To whom requests for reprints should be addressed.

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v/v), HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethane-sulfonic acid]; 10 mM, pH 7.2), penicillin (100 U/ml), and streptomycin (100 #g/ml); (all materials for this medium were ob= tained from Gibco Life Technologies, Grand Island, NY). Under aseptic conditions, the neurohypophysis was isolated by removal of the anterior and intermediate lobes of the pituitary and cut into four approximately equal sized pieces. Each piece of tissue or explant was placed on a separate sterile plastic coverslip (22 mm diameter, Lux Thermanox; Miles Laboratories, Naperville, IL) and attached to the coverslip by a clot formed from chicken plasma (0.5 mg/ml; Sigma Chemical Co, St Louis, MO) and bovine thrombin (10.8 U/ml). Cultures were maintained in culture medium in a humidified atmosphere (37°C) containing 95% air and 5% CO2; the culture medium was replaced after 7 days.

Exposure of Pituicytes to Experimental Media

FIG. 1. Photomicrographs of hematoxylin-stained pituicytes and of pituicytes immunocytochemically stained for glial fibrillary acidic protein (GFAP). (Ai) Cultured cells from the adult rat neurohypophysis appear mostly nonstellate in culture medium and when incubated in HEPES buffered salt solution (HBSS) alone; (Aii) cultures incubated in forskolin- or isoproterenol-supplemented HBSS contain many stellate cells (Scale bar represents 100 #m.) (B) Stellate cells observed (i) under phase contrast and (ii) under epifluorescence were identified as pituicytes because they were immunoreactive to antiserum against GFAP. (C) Nonstellate cells observed (i) under phase contrast and (ii) under epifluorescence were also identified as pituicytes by their immunoreactivity to antiserum against GFAP. (D) Nonstellate cells observed (i) under phase contrast and (ii) under epittuorescence were not stained when the immunocytochemical procedure was performed without the primary antiserum against GFAP. (Scale bar in Dii represents 100/~m for B, C and D.) induced by forskolin is substantially and significantly reduced when serum is included in the forskolin-supplemented medium. MATERIALS AND METHODS

Culture of Pituicytes The methods that we used to culture pituicytes follow those first described by Bicknell and colleagues [2]. Young adult male S p r a g u e - D a w l e y rats ( 1 7 5 - 2 0 0 g; Harlan) were euthanized by decapitation. The pituitary gland was removed and placed into sterile culture medium, which consisted of Dulbecco's Modified Eagle Medium supplemented with newborn calf serum (10%

After 14 days in culture, the explant-containing clot in each culture was removed from the coverslip to leave a monolayer of cells, which had grown out of the explant, adhered to the coverslip. The cultures were then rinsed three times with a newborn calf serum (0.05% v/v) supplemented HEPES buffered salt solution (HBSS), which contained (raM) HEPES 10, NaC1 150, KC15, CaC12 2, MgC12 2, and D-glucose 5.6, (pH 7.4, 310 mOsm/ kg). Experimental medium (HBSS, with supplements, including isoproterenol and forskolin, as required) was then added to each dish, and the cultures were incubated for 90 rain (37°C, 95% O~ and 5% CO2 atmosphere); (isoproterenol was obtained from Sigma Chemical Co, and forskolin was obtained from Research Biochemicals International, Natick, MA). Following this treatment, the cultures were either (i) fixed with paraformaldehyde (4%) and picric acid (0.2%) in phosphate buffer (0.15 M, pH 7.6) and then stored in HBSS (at 4°C), or (ii) incubated in an experimental medium for another 90-min period and then fixed. Experimental media were letter coded immediately prior to use so that treatment of the cultures could be performed " b l i n d . " After fixation, culture dishes were also coded so that the subsequent microscopic examination of the cultures could be performed " b l i n d " to treatment group assignment.

Assessment of Pituicyte Morphology The morphology of the pituicytes was examined with phase contrast optics. Cells were judged as being stellate if they possessed a clearly defined cell body with one or more distinct processes; nonstellate cells had neither a well-defined soma nor distinct processes (see Fig. 1). In each culture, four separate fields of cells (1 mm ~ area) at the outer edge of pituicyte outgrowth were examined. In each field, the numbers of stellate and nonstellate cells were determined, and the counts for all four fields in a single culture were added together. For each culture, the ratio of the number of stellate cells to the total number of stellate and nonstellate cells was calculated to give the fraction of stellate cells within that culture. For each treatment group within an experiment, 3 - 4 cultures were treated with each experimental medium or sequence of two experimental media in each of two or three replicates. Therefore, in each experiment, each treatment group consisted of 8 - 1 2 cultures: in the figures presented here, the mean and standard error of the mean for each treatment group within an experiment is shown. The M a n n - W h i t n e y U-test was used for detection of statistically significant differences between groups treated with the different media that we used.

lmmunocytochemical Procedures To demonstrate that the cells that exhibited morphological plasticity in our cultures were indeed astrocytic glia, and thus

S E R U M A N D PITUICYTE PLASTICITY

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Treatment FIG. 2. Isoproterenol induced stellation of pituicytes is not reversed by isoproterenol removal. The fraction of stellate cells was significantly increased when pituicytes were incubated in HEPES buffered salt solution (HBSS) supplemented with isoproterenol ( ' T ' ) compared to HBSS alone ( " C " ) for 90 min. The fraction of stellate cells remained low when pituicytes were incubated in HBSS alone for two 90 min periods ("C/C"), and stellation was significantly increased by incubation in isoproterenol supplemented HBSS (90 min) immediately after incubation in HBSS alone (90 min; "C/I"). Stellation induced by incubation in isoproterenol supplemented HBSS (90 min) was not reversed when pituicytes were further incubated (90 min) in isoproterenol supplemented HBSS ("I/I") or HBSS alone ("I/C"). (*significantly different to " C " ; Mann-Whitney U-test, p < 0.05.). pituicytes, some cultures were stained immunocytochemically using primary antiserum against the astrocytic marker glial fibrillary acidic protein (GFAP). Cultures were first incubated (at 21°C) in goat serum (2%, 120 min) to prevent nonspecific staining of cells by the fluorescent labelled secondary antiserum. Then cultures were incubated in polyclonal primary antibody against G F A P (raised in rabbit, used at 1:400; Chemicon International Inc., Temecula, CA) for 48 h at 4°C. After rinsing in phosphate buffered saline the cultures were then incubated in Cyanine 3 labelled secondary antiserum (goat antirabbit IgG, 1:1000; Jackson Immunochemical Labs, West Grove, PA) for 2 h at 21°C, and, finally, rinsed and mounted in Tris buffered (pH 8.6) glycerol for observation using phase contrast and epifluorescence optics. RESULTS Cells at the outer edge of cell growth from the neurohypophysial explants were mostly nonstellate after incubation in HBSS alone (Fig. 1A) but mostly stellate after incubation in HBSS containing forskolin (Fig. 1B) or isoproterenol. Most ( > 95%) of the cells in these neurohypophysial explant cultures were identified as being pituicytes because they were positively immunocytochemically stained using antisera against glial fibrillary acidic protein (Fig. 1B,C,D). In the first study to examine reversal of the stellation response, we determined whether pituicytes that were first induced to become stellate would revert to the nonstellate form after the stellation-inducing stimulus was removed. Figure 2 shows that there was a significantly higher fraction of stellate cells in cultures incubated in HBSS containing isoproterenol (90 min) than in

HBSS alone (90 rain or 180 min) and that the effect of isoproterenol was observed even if the pituicytes were incubated in HBSS alone (90 min) immediately prior to being incubated in HBSS supplemented with isoproterenol (90 min). These observations show that the pituicytes are capable of responding to isoproterenol even after being incubated in serum-free medium, that is, HBSS, for 90 min, and that 180 min incubation in HBSS alone did not induce stellation to occur. The stellation response induced by isoproterenol was also maintained if the pituicytes were incubated in HBSS supplemented with isoproterenol for two consecutive 90 min periods. The latter demonstrates that stellation induced by exposure to isoproterenol is not a transient response, and therefore if reversion to the nonstellate form occurred after removal of isoproterenol, then reversion must be due to removal of the stellation-inducing stimulus and could not be due to an inability of the cytoskeleton to maintain the stellate form. Pituicytes incubated first in HBSS containing isoproterenol (90 min) and then in HBSS alone (90 min) did not revert to the nonstellate form (Fig. 2), suggesting that simply removing the stimulus to stellation does not cause the pituicytes to revert to their original nonstellate form. In the second study to examine reversal of the stellation response, cultures were incubated (90 min) in HBSS alone and then fixed (pituicytes in these cultures were expected to remain nonstellate) or were incubated (90 min) in HBSS supplemented with forskolin (to induce stellation). Cultures exposed to forskolin were immediately fixed or were transferred immediately to HBSS supplemented with 0.5% newborn calf serum or to HBSS alone for a second 90 min incubation period before being fixed. The fraction of stellate cells in cultures incubated in HBSS supple-

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mented with forskolin (90 min) was significantly greater than in cultures incubated in HBSS alone (90 min) as expected (Fig. 3). W h e n forskolin exposed, stellate pituicytes were transferred to HBSS containing serum (90 min) but not to HBSS alone (90 rain), there was a significant reduction in the fraction of stellate cells (Fig. 3). In fact, in cultures incubated first in HBSS supplemented with forskolin and then in HBSS supplemented with serum, there was significantly lower fraction of stellate cells than observed in cultures incubated in HBSS alone for 90 min (Fig. 3). Because it was clear that inclusion of serum in the incubation medium induced reversion of stellate pituicytes to the nonstellate form within 90 rain, the effect of serum on the induction of stellation was examined. Cultures incubated in HBSS supplemented with forskolin had a significantly greater fraction of stellate cells than when incubated in HBSS alone, as expected (Fig. 4). The response to forskolin was substantially attenuated, although not totally blocked when cultures were incubated in HBSS supplemented with forskolin and serum (Fig. 4), W e also observed that the fraction of cells that was stellate was significantly higher when cultures were incubated in HBSS alone than when incubated in HBSS supplemented only with serum. DISCUSSION Previous studies of the role of serum or serum components to control the morphology of glioma-derived astrocytes have focussed on the reversal of morphological changes induced by elevation of intracellular cyclic A M P following activation o f / 3 adrenoreceptors [9,20]. Reversion to the nonstellate form takes

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FIG. 4. Serum reduces forskolin-induced morphological changes exhibited by cultured pituicytes. Incubation (90 min) of cultures in HEPES buffered salt solution (HBSS) supplemented with forskolin ( " F " ) induced a significant increase in the fraction of stellate cells compared to incubation in HBSS alone ("C"). The effect of forskolin was reduced when HBSS was supplemented with forskolin and new born calf serum ( " F + S " ) . Cultures incubated in HBSS supplemented only with serum ( " S " ) had a significantly lower fraction of stellate cells than in HBSS alone. (*significantly different to "C";-significantly different to " F " ; Mann-Whitney U-test, p < 0.05.)

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FIG. 3. Serum reverses forskolin-induced stellation of cultured pituicytes. Incubation (90 min) of pituicyte cultures in HEPES buffered salt solution (HBSS) supplemented with forskolin ( " F " ) significantly increased the fraction of cells that was stellate compared to incubation in HBSS alone ("C"). The effect of incubation of cultures in incubation in HBSS Supplemented with forskolin ( " F " ) was reversed when stellate pituicytes were incubated (90 min) in HBSS supplemented with newborn calf serum ("F/S") but not when incubated in HBSS alone for the same period ("F/C' '). (*significantly different to "C";-significantly different to " F " ; Mann-Whitney U-test, p < 0.05.)

place in these glioma-derived cells more rapidly when serum or two of its components (thrombin and lysophosphatidic acid) is present than in serum-free conditions. Here we have shown that primary cultured pituicytes from the neurohypophyses of adult rats behave similarly to glioma-derived cells. It should be noted that the change from the stellate form to the nonstellate form appears to be a real reversal of the stellation process and not simply a rounding up of the cells by process withdrawal as occurs in some differentiated neuroblastoma-derived cells when exposed to serum or thrombin [3,8]. Activation of/3-adrenoreceptors or exposure to forskolin induces pituicytes to become stellate through a presumed cyclic AMP-dependent mechanism, and reversal of this process after removal of the stellation inducing stimulus occurs more rapidly when serum is added to the incubation medium than when serum is absent. Although lysophosphatidic acid and thrombin not only induced reversal of stellation and also induced a transient increase in free intracellular Ca 2+ concentration in glioma-derived astrocytes, other agents that produce a similar change of intracellular Ca 2+ concentration did not induce reversal of stellation [9]. Koschel and Tas [9] concluded that the PIP2 cleavage system is not involved in the morphological change induced by serum (components). An alternative mechanism for the effect of serum (components) is that serum (components) induce a reduction of c A M P concentration through an action on adenylate cyclase, as has been demonstrated for fibroblasts [ 12,28]. Although serum may induce a rapid reduction of intracellular cyclic A M P and reversal of the stellation process in glioma-derived cells through activation of an inhibitor G-protein linked to adenylate cyclase, we do not believe that this mechanism is significantly involved in reversal

SERUM AND PITUICYTE PLASTICITY

of stellation exhibited by pituicytes in our experiments for two reasons. First, in our study, the potential for pituicytes to undergo reversal of stellation was examined after removal of the stellation-inducing stimulus (isoproterenol or forskolin), and thus adenylate cyclase activity would be at a basal level when exposure o f stellate pituicytes to HBSS supplemented with serum was initiated. Second, if G-protein inhibition of adenylate cyclase is the mechanism by which serum induces reversal of stellation in pituicytes, then the basal level of adenylate cyclase activity must be high if a high cyclic A M P concentration must be maintained to sustain the stellate morphology. That basal activity of adenylate cyclase is high enough to sustain the stellate form is unlikely because high basal activity would induce nonstellate pituicytes to become stellate when incubated in HBSS alone, an event that does not occur. Because pituicytes do b e c o m e stellate when incubated in HBSS supplemented with the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX; our unpublished observations), basal phosphodiesterase activity must be high. Therefore, removal of the stellation-inducing stimulus might be expected to be followed by a rapid decline of cyclic A M P concentration even in the absence of serum. Although we have not measured cyclic A M P concentration in pituicytes, we believe that reversal of the stellation process can occur without a decrease of intracellular cyclic A M P concentration. Rather, we hypothesize that serum may induce reversal of the stellation process in a manner independent of intracellular cyclic A M P concentration by activating a protein phosphatase so that cytoskeletal proteins are dephosphorylated and the stellation process is reversed. In all previous studies of morphological plasticity of cultured astrocytes or astrocyte-like cells, serum was absent from the solutions used to test whether transmitters and hormones can induce stellation [7,13-18,29] or serum was added to the solution at a low concentration [2,6], and the effects of serum on the stellation process have not previously been investigated. The m e c h a n i s m by which serum inhibits stellation is unknown. Serum may act to inhibit adenylate cyclase through a receptor-activated inhibitory G protein [12,28], or serum may activate cyclic A M P phosp h o d i e s t e r a s e - - e i t h e r of these processes would prevent the increase of intracellular cyclic A M P concentration that is necessary for stellation. Alternatively, serum may inhibit stellation by producing instability of the cytoskeletal elements necessary to produce and maintain the stellate morphology or by activating a protein phosphatase to prevent the changes in the cytoskeleton that underlie stellation. Although our present experiments demonstrate that serum affects the morphology of cultured pituicytes in culture, the role o f serum in controlling neurohypophysial neuroglial relations in vivo is unknown. It is noteworthy that the neurohypophysis is a circumventricular organ, and the capillaries within the neurohypophysis are fenestrated so that serum components can therefore cross into the perivascular space from the blood and contact the pituicytes. W e suggest that the ultrastructural changes in the neurohypophysis in vivo induced when oxytocin and/or vasopressin secretion from the neurohypophysis has been stimulated is attenuated by serum components, which affect pituicyte morphology. This hypothesis would offer an explanation to the observation that detection of ultrastructural changes within the hypothalamo neurohypophysial system during sustained elevated hormone secretion requires several hours following the onset of the stimulus to elevate hormone secretion [ 1,22]. This hypothesis is also supported by the observations that rapid morphological changes may be induced in the neurohypophysis in serum-free conditions in vitro [10,12,19] and in the supraoptic nucleus in situ during per-

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fusion of the brain with hypertonic serum-free medium immediately post mortem [27]. In conclusion, we have shown that serum has significant effects on the plasticity of the morphology of cultured pituicytes induced by agents that elevate intracellular cyclic AMP. The underlying m e c h a n i s m of these serum-induced effects and their relevance to events that occur in vivo as hormone secretion from the neurohypophysis increases and decreases as physiological conditions changes are presently under investigation. ACKNOWLEDGEMENTS This work was supported by a FIRST award to P.C. from NINDS (NS28206) and by an internship to D.L.B. from The American Society of Pharmacology and Experimental Therapeutics. The authors thank Dr. Diane Matesic for her critical readings of the manuscript.

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