Morphological plasticity and rearrangement of cytoskeletons in pituicytes cultured from adult rat neurohypophysis
Introduction
The pituicytes, modified astrocytes are the predominant cellular type in the neurohypophysis, and have an intimate but plastic relationship with axonal terminals of the neurohypophysial hormones, oxytocin and vasopressin, the so-called morphological plasticity (Hatton, 1990, Hatton, 1997). In unstimulated conditions, processes of pituicytes are located as interposing between axonal terminals and basal lamina surrounding the capillary vessels (Tweedle and Hatton, 1980, Tweedle and Hatton, 1987). However, pituicytes appear to retract from the axonal terminals during chronic physiological stimulation such as dehydration and lactation, so that ratio of the axonal terminals in direct contact with the basal lamina increases (Tweedle and Hatton, 1980, Tweedle and Hatton, 1987, Luckman and Bicknell, 1990). The reasons why this structural reconstitution permits to enhance the release of neurohypophysial hormones are as follows; First, retraction of pituicyte process at the basal lamina results in removal of a diffusion barrier, so that released neurohypophysial hormones reach into blood vesseles more efficiently (Tweedle and Hatton, 1980, Tweedle and Hatton, 1987). Secondly, an increase in extracellular K+ level enhances hormonal release from the axonal terminal as seen in GnRH neuron (Witkin et al., 1991). Finally, a reduction of glial coverage of the axonal terminals leads to facilitate the diffusion of neurotransmitters and/or neuromodulators, and then changes the activity of neighboring axons and glia in a paracrine fashion (Boersma and Leeuwen, 1994, Miyata et al., 1997). Thus, it is well established that pituicyte shape conversion largely contributes to drastic and reversible morphological plasticity in the adult neurohypophysis with chronic physiological stimulation. Therefore, investigation of the mechanism for shape conversion of the pituicyte morphology in the neurohypophysis is very important for understanding morphological plasticity of adult brains under physiological conditions.
Pituicyte shape conversion has also been found in the isolated whole neurohypophysis; isoproterenol (IPR; β-adrenergic agonist induces ultrastructural changes which are quite similar to those seen in the neurohypophysis of dehydrated or lactating animals (Luckman and Bicknell, 1990, Smithson et al., 1990). Lafarga et al. (1992) have demonstrated that hypothalamic astrocytes of neurohypophysial neurons increase their expression of β-adrenergic receptor in response to dehydration. Pituicyte shape conversion is also mimicked in cultured pituicytes from the neurohypophysis explants of adult rat (Bicknell et al., 1989, Hatton et al., 1991). β-adrenergic agonist changes the morphology of cultured pituicyte from flat to stellate shape (stellation). Stellation of cultured pituicyte is mediated through cAMP-dependent process, because treatment with forskolin, an activator of adenylate cyclase, and 8-bromo cAMP, a permeable cAMP analog, causes stellation of the pituicyte morphology (Zhao and Cobbett, 1993, Ramsell and Cobbett, 1997). It is also shown that serum and nitric oxide is shown to be concerned with shape conversion of cultured pituicyte (Ramsell and Cobbett, 1996, Ramsell et al., 1996, Ramsell and Cobbett, 1997).
In adult brains, astrocyte shape conversion is seen when brains are damaged by hypoxia, ischemia, seizures (Nathaniel and Nathaniel, 1981, Mathewson and Berry, 1985, Norton et al., 1992, Eng and Ghirnikar, 1994), Alzheimer’s disease, or Down syndrome (Jorgensen et al., 1990). These so-called activated astrocytes have more prominent processes with more branching than normal astrocytes (Shao and McCarthy, 1994). Astrocyte shape conversion plays a role in guiding migration and axonal growth of neurons during embryonic development (Mason et al., 1988). Until now, there have been so many reports investigating the mechanism for shape conversion of brain astrocytes cultured from neonatal brains (Shain et al., 1992, Shao and McCarthy, 1994, Baba, 1998). But, little is known whether the mechanism of pituicyte shape conversion is same to that of brain astrocyte, although pituicyte shape conversion is induced with physiological demands rather than brain damages or development. Then, the present experiments aimed to investigate the receptor mechanism for pituicyte shape conversion and functional role of cyotoskeletons on shape conversion of cultured pituicytes from adult rat neurohypophysis. Quite recently, we have demonstrated using western blot and dual labeling immunohistochemistry that pituicytes in the neurohypophysis of adult rats express low-molecular-weight form of microtubule-associated protein-2 (LMW MAP2) (Matsunaga et al., 1999). The presence of immature variant, LMW MAP2 presumes that rearrangement of microtubules is required for shape conversion of the pituicyte morphology during chronic physiological stimulation.
Section snippets
Pituicyte culture
The pituicyte culture was performed with a slightly modified method of previous studies (Bicknell et al., 1989, Hatton et al., 1991). Briefly, the Wistar males (6–8 weeks) were killed by decapitation and the pituitary glands were immediately removed. The neurohypophysis was dissected free from the anterior and intermediate pituitaries in sterile culture medium (Dulbecco’s modified Eagle/F12) under a dissecting microscope. The neurohypophysis was then cut into four pieces and each piece was
Effects of various bioactive substances on pituicyte shape conversion
In normal culture medium, pituicytes were flat amorphous shape. After incubation in the HBSS, most of the control pituicytes at the outer edge of cell growth from the neurohypophysis explants remained flat shape (Fig. 1A and C). Treatment with 100 nM IPR (Fig. 1B) and 1 mM dBcAMP (Fig. 1D) for 60 min induced shape conversion of the pituicyte morphology, from flat to stellate shape (stellation). Adenosine (more than 1 μM) also induced stellation of the pituicyte morphology (Table 1). The
Discussion
In the present study, stellation of the pituicyte morphology was induced by IPR and adenosine, and ET-1 and ET-3 caused spreading. Various kinase inhibitors (sphingosine, W-7 and ML-9) and CytB also induced Stellation. Stellation with dBcAMP treatment was prevented by pretreatment with colchicine and tyrosine phosphate inhibitors (orthovanadate and phenylarsine). Moreover, rapid rearrangement of microfilaments and microtubules was observed during shape conversion of the pituicyte morphology
Acknowledgements
This work was supported in part by grants for Scientific Research from Ministry of Education, Science, Sports and Culture of Japan and the Salt Science Research Foundation.
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