Role of endothelin b receptor signals in reactive astrocytes

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ROLE OF ENDOTHELIN

PII MOM-3205(98)00133-7

Life Sciences, Vol. 62, Nos. 17/l& pp. 1711-1715, 1998 copyright 0 1998 Eamier science Inc. Printed in the USA. All rights reserved Mm-3205/98 $19.00 t .tm

B RECEPTOR SIGNALS IN REACTIVE ASTROCYTES Akemichi Baba

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Yamada-oka, Suita, Osaka 565, Japan

l-6

Summary We investigated the involvement of endothelin, receptor signals in the activation of astrocytes in vitro and in vivo. Endothelin reversed the stellate morphology of astrocytes induced by several agents. Endothelin stimulaltes astrocytic stress fiber formation. This effect of endothelin is Ca*‘-independent and mediated by rho protein signal cascades linked to tyrosine phosphorylation. Injection of endothelin into striatum caused reactive astrocytosis which is prevented by a systemic injection of endothelin, receptor antagonist. We propose the role of endothelin, receptor signal transduction in reactive astrocytes. Key Wordr: endothelin, astrocytes, actin, rho

Endothelins (ETs) are present in high levels in brain and show neurotransmitter- or modulator-like actions on nervous tissues. Because the production of ETs is stimulated at injured brain sites (1, 2) ETs are suggested to have some pathophysiological roles in brain injuries. Receptors for ETs An in situ study of rat brain showed that ET, have been classified into ET, and ET, subtypes. and ET, receptors were differently distributed among cell types (3). ET, receptors are expressed in brain vascular cells, whereas ET, receptors are expressed predominantly in glial cells of brain (3). Astrocytes have receptors for ETs and are thought to be target cells for brain ETs. Activation of astrocytic ET receptors induces increases in [Ca”], , phosphatidylinositol breakdown, and attenuation of CAMP formation (4-7). These signals are involved in the stimulation of However, little is known about the mitosis and gene expression in astrocytes. In the following, I described the role of pathophysiological roles of ETs on astrocytic functions. ET, receptor signals on astrocytic morphology and the phenotypic changes into reactive astrocytes. Regulation of astrocytic morphology and cytoskeletal organization by ETs Morphology of cultured astrocytes is changed by several agents and in different culture conditions. Treatment with 1 mM dibutyryl (DB) CAMP, 10 mM forskolin, 100 mM isoproterenol and 500 nM phorbol 12-myristate 13-acetate changed protoplasmic cultured astrocytes into process-bearing ET-3 (1 nM) completely prevented the astrocytic stellation induced by these agents (IF,,, ones. 49 PM’). Furthermore, stellate astrocytes were reversed to the protoplasmic type cells by addrtton of 1 nM ET-3 in the presence of DBcAMP (8). ET-3 reversed the astrocytic stellation in the Preloading of BAPTA-AM, a permeable Ca” chelator, on stellate absence of extracellular Ca” astrocytes had no effect on the reversal by ET-3. ET-3 did not increase intracellular free Ca” concentrations ([Ca”],) of most astrocytes tested at 0.1 nM. These results suggest that ETs modulate morphological changes in astrocytes through CAMP- and Ca*‘-independent mechanisms (8). Protoplasmic type cultured astrocytes (type 1 astrocytes) change their morphology to a process-bearing shape by treatment with CAMP analogues and hormones activating adenylate Reorganization of the cytoskeleton underlies some cellular functions, including cyclase. In serum-free medium, maintenance of the morphology, locomotor activity, mitosis and cytosis. treatment with DBcAMP causes reversible cytoplasmic retraction of astrocytes in a few hours.

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Such morphological changes induced by DBcAMP are accompanied by depolymerization of actin filaments, while microtubules and intermediate filaments remain intact during the treatment (9) suggesting that the cytoplasmic retraction of cultured astrocytes is accompanied by reorganization of actin filaments. In serum-free medium, treatment with cytochalasin B, an inhibitor of actin polymerization, caused astrocytic morphological changes with stellation which is prevented by a simultaneous addition of ET-l, ET-3 and sarafotoxin S6b (10). Similarly to the case of DBcAMP-treated astrocytes, ET-3 reversed the stellate morphology of cytochalasin B-treated cells; the cells lost their apparent distinction between cell body and processes 60 min after the addition of 1 nM ET-3. Treatment with DBcAMP and cytochalasin B decreased the content of actin in the cytoskeletal fraction by about 30%. Subsequent addition of 1 nM ET-3 for 2 hr restored the decreased actin content to that of control cells (10). Possible changes of astrocytic actin organization was, therefore, examined by rhodamine-phalloidin staining of the cell. Protoplasmic astrocytes showed a prominent structure of organized filamentous actin, the stress fiber. The astrocytic stress fibers disappeared after treatment with DBcAMP and cytochalasin B. ET-3 stimulated reorganization of stress fibers both in the DBcAMP- and the cytochalasin B-treated astrocytes (10). Although, stress fibers and reversal of cytoplasmic retraction were observed after ET-3 addition, the morphology of cytochalasin B-treated astrocytes differed from that of nontreated astrocytes. These results show that ET-3 stabilizes or polymerizes cytoskeletal F-actin against cytochalasin B and DBcAMP, but it is not clear whether stress fiber formation by ET-3 is produced by conversion between globular actin and F-actin, or by rearrangement of F-actin organization. Signals involved in ET, receptor -mediated actin-organization in astrocytes As described above, the ET-induced expansion of astrocytic cytoplasm was accompanied by formation of stress fibers (lo), an organized cytoskeletal actin structure, and the effect on astrocytic morphology was independent of changes in CAMP and Ca2+ (8). Phosphorylation of cytoskeletal proteins by CAMP-dependent protein kinase is responsible for morphological changes in non-muscle cells. Phosphorylation of myosin light chain kinase, an actin depolymerizing factor and glial Iibrillary acidic protein (GFAP) are suggested to be involved in regulation of astrocytic stellation (11,12), suggesting that another signal underlies the novel action of ETs. We investigated signal transduction mechanisms of ET receptor-mediated actin reorganization of rat cultured astrocytes (TABLE I&II). Pretreatment with 0.1 &ml pertussis toxin (PTX) and chelation of cytosolic Ca” did not affect astrocytic stress fiber formation by ET-3. ET-3 stimulated stress fiber formation in stellate astrocytes induced by 50 uM ML-9, 20 uM W-7, and 5 PM cytochalasin B (13). In addition 100 uM phenylephrine, an CL,agonist, 1 PM bradykinin, and 100 nM phorbol 12-myristate did not stimulate stress fiber formation in DBcAMP-induced stellate astrocytes. Although an increase in cytosolic Ca” is responsible for many actions of ETs, these observations indicated that Ca” is not involved in the action of ET-3. In addition to ET, receptors, a, and bradykinin receptors arc coupled to PTX-insensitive G proteins and activate PKC systems (14-16). The lack of effect of phenylephrine, bradykinin, and PMA on stress fiber formation shows that PLC-PKC system is not a major pathway for astrocytic actin organization. Recent studies show that rho proteins, a family of ras-like small G proteins, transduce receptor signals to stress fiber formation (17-19). C, ADP-ribosyltransferase produced by C. botulinum (C, enzyme) impairs interactions of rho protems with the effecters (20) and thus is used to reveal rho-protein-mediated cellular responses. We found that only 21-23 kDa proteins of astrocytic homogenate were ribosylated by C, enzyme, and the ribosylated proteins had rho A immunoreactivity. Microinjection of C, enzyme into DBcAMP- and cytokalasin B-induced stellate astrocytes completely prevented the effect of ETs on stress fiber formation. C, enzyme did not affect increase in cytosolic Ca2’ by ET-3, indicating that the site of the action of C, enzyme is not on ET receprors. From these results, rho proteins are suggested to be involved in ET-induced astrocytic actin reorganization (13).

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TABLE I Effects of agents on intracellular Ca2+ and stress fiber formation in cultured astrocytes

Agents

Intracellular Ca*+

ETs (cl nM) (>l nM) phenylephrine

increase increase

bradykinin phorbol 12-myristate

Stress fiber formation t t

increase increase

In addition, in accordance with the previous finding (20) we confirmed that ML9 and W-7 caused stellation and the disappearance of stress fibers in cultured astrocytes, indicating a regulation of stress fiber formation by myosin light chain kinase (MLCK) and calmodulin. However, the possible involvement of MLCK in ET-induced actin reorganization is excluded by the observations that ET-3 reversed the effects of ML9 and W-7. It is reasonable that signal transduction pathways other than MLCK regulate stress fiber formation down-stream of rho proteins (13).

TABLE II Specificity of endothelin-induced

Agents

reversal of stellate morphology of astrocytes

Stellation

Endothelin-reversal

DBcAMP

+

+

cytochalasin B ML9

+ +

t t

w-7

+

t

Promotion of astrocytic activation by endothelins in vivo Multiple types of cells are involved in the tissue repair process of damaged brain. In various brain damages including Alzheimer’s disease, AIDS dementia and acute traumatic brain injuries, The reactive astrocytes are developed by a activation of astrocytes is commonly observed. phenotypic conversion of resting astrocytes, which are characterized by hypertrophy of cell body At the injured sites, reactive astrocytes often and processes, and by a high expression of GFAP. replace the degenerated neurons and form glial scars, which leads to the hypothesis that glial scars, In contrast, by acting as a physical barrier, prevent axonal elongation of remaining neurons. reactive astrocytes produce neurotrophic substances and cell adhesion molecules, which leads to the hypothesis that the astrocyte-derived molecules reduce neuronal damage and stimulate axonal elongation . Our previous

in vitro observations (8,10,13) suggest modulation of astrocytic functions by ETs We, therefore, examined the effect of ET on through rho protein-mediated signal cascade. In that experiment, ET, receptor agonists were injected into astrocytic activation in vivo (21). and the subsequent astrocytic activation was examined by an rat caudate putamen, One week after a single injection of ET-3 (40 pmole), imrnunohistochemical staining of GFAP.

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CAMP

Ca’+ 1

, rh y

Lrl PKC

e phospho y

1

\ Cytoskeleta ) organization \

Astrocytic Fig. 1.

Gene expression Proliferation

+------

activation

ET, receptor signal transduction pathways leading to astrocytic activation

GFAP-positive cells in the caudate putamen increased compared to that in the contralateral side. -ET-l, a selective ET, receptor agonist, Similarly, a single injection of 40 pm01 Ala1.3,11.1s Time-course study showed that the increased GFAP immunoreactivity in the caudate putamen. number of GFAP positive cells increased gradually in the first week, reached maximum (about 50% increase in number) in l-2 weeks and disappeared in 4-8 weeks after ET injection. A continuous infusion of BQ788, a selective antagonist for ET, receptor (22), into the lateral ventricle of the cerebrum significantly decreased GFAP-positive cell number in the ET, receptor In addition, it should be noted that BQ788 also decreased the agonist-injected caudate putamen. number of GFAP-positive cells in the control side. In these in vivo experiment, it is necessary to examine whether the increase in number of reactive astrocytes is directly induced by ET. Activated microglia secrete factors modulating astrocytic activation (23). Microglia 1 cells aggregating at injured sites possessed ET, receptors (24). This raised the possibility that the ET, receptor agonist promoted astrocytic activation by activating Therefore, the activation of brain microglias was monitored by using B, microglial cells. The number of B,-positive cells increased for 1 week and rapidly decreased thereafter, isolectin. and the ET, receptor agonist did not increase further the number of B,-positive cells for 7 weeks (21). Degeneration of neurons is one of the prime events preceding astrocytic activation, but the Nissl-staining and immunochemical staining of neurofilaments of caudate putamen indicated that injections of ETs did not cause degeneration of striatal cells, including neurons (21). These observations suggest that ETs promote selectively the activation of astrocytes but not microglia and neurons. Roles of astrocytic ET, receptors in activation of astrocytes ETs induced stress fiber formation of cultured astrocytes in vitro and increased the number of GFAP-positive cells in vivo through activation of ET, receptors, because the potencies of ETs for these effects were similar and prevented by BQ788 (8,13,21). As shown in Fig. 1, astrocytic ET, receptors are linked to several signal cascades. In our previous observation, we found that ETS at low concentrations, in which they did not increase cytosolic Ca”, stimulated tyrosine phosphorylation of cytoskeletal proteins of astrocytes. Those proteins are immunologically The observations indicate that ETs defined as focal adhesion kinase and paxillin (not published). The involvement of stimulated tyrosine phosphorylation of focal adhesion proteins of astrocytes.

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tyrosine phosphorylation of cytoskeletal proteins in stress fiber formation was also supported by the finding that vanadate, an inhibitor of protein tyrosine phosphatase, induced astrocytic stress fiber formation (25). In addition, C, ADP ribosyltransferase blocked stress fiber formation induced by ETs and vanadate (13,2.5), indicating an involvement of rho proteins down-stream of ET, receptor signals leading to astrocytic cytoskeletal organization. Since activation of astrocytes in vivo results in phenotypic changes of the cells, the activation is composed of several biological processes, such as gene expression and secretion of active substances, proliferation and cellular activities including adhesion and phagocytosis. Many endogenous substances are responsible for these cellular activities of astrocytes. Our findings clearly indicate that ETs are one of the endogenous substances responsible for the production of reactive astrocytes during brain injuries.

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