Review

Mechanosensation and endothelin in astrocytes—hypothetical roles

in CNS pathophysiology

Lyle W. Ostrow, Frederick Sachs*

Department of Physiology and Biophysics, S.U.N.Y. at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, NY 14214, USA

Accepted 9 September 2004

Available online 28 October 2004

Brain Research Reviews 48 (2005) 488–508

www.elsevier.com/locate/brainresrev

Abstract

Endothelin (ET) is a potent autocrine mitogen produced by reactive and neoplastic astrocytes. ET has been implicated in the

induction of astrocyte proliferation and other transformations engendered by brain pathology, and in promoting the malignant behavior

of astrocytomas. Reactive astrocytes containing ET are found in the periphery/penumbra of a wide array of CNS pathologies.

Virtually all brain pathology deforms the surrounding parenchyma, either by direct mass effect or edema. Mechanical stress is a well

established stimulus for ET production and release by other cell types, but has not been well studied in the brain. However,

numerous studies have illustrated that astrocytes can sense mechanical stress and translate it into chemical messages. Furthermore, the

ubiquitous reticular meshwork formed by interconnected astrocytes provides an ideal morphology for sensing and responding to

mechanical disturbances. We have recently demonstrated stretch-induced ET production by astrocytes in vitro. Inspired by this finding,

the purpose of this article is to review the literature on (1) astrocyte mechanosensation, and (2) the endothelin system in astrocytes,

and to consider the hypothesis that mechanical induction of the ET system may influence astrocyte functioning in CNS

pathophysiology. We conclude by discussing evidence supporting future investigations to determine whether specific inhibition of

stretch-activated ion channels may represent a novel strategy for treating or preventing CNS disturbances, as well as the relevance to

astrocyte-derived tumors.

D 2004 Elsevier B.V. All rights reserved.

Theme: Development and regeneration

Theme: Glia and other non-neuronal cells

Keywords: Stretch; Calcium; Proliferation; Glioma; Channel blocker; Cytoskeleton; GFAP

Contents

1. The temperament of astrocytes: structural support for neurons or active role in CNS functioning? . . . . . . . . . . . . . . . 489

2. Astrocyte responses to CNS disturbances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

3. Brain deformation and pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490

4. Astrocyte mechanosensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490

4.1. Stretch-induced Ca2+ waves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490

4.2. Mechanosensitive ion channels (MSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

4.3. Cytoskeletal remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

4.4. Mechanosensation is dynamic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

0165-0173/$ - s

doi:10.1016/j.br

* Correspon

USA. Fax: +1 7

E-mail addr

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ee front matter D 2004 Elsevier B.V. All rights reserved.

ainresrev.2004.09.005

ding author. Hughes Center for Single Molecule Biophysics, Physiology and Biophysical Sciences, 320 Cary Hall, SUNY, Buffalo, NY 14214,

16 829 2569.

ess: sachs@buffalo.edu (F. Sachs).

www.sachslab.buffalo.edu.

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508 489

5. Endothelins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495

5.1. Endothelin in astrocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495

5.2. Endothelin regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496

6. Potential therapeutic targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

6.1. ET-receptor antagonists as treatments for brain injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

6.2. Inhibitors of stretch-activated ion channels (SACs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

7. Mechanical deformation and endothelin in astrocyte-derived tumors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499

8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501

1. The temperament of astrocytes: structural support for

neurons or active role in CNS functioning?

Astrocytes, or astroglia, along with micro- and oligoden-

dro-glia, make up the major non-neuronal cellular compo-

nents of the central nervous system (CNS). Since they are

not neurons, they were historically considered to play

mainly a structural role. In fact, the word glia comes from

the Greek for bglue.Q The notion of a purely physical role forastrocytes evolved as a result of their distinctive topology—

impressively stellate cells forming an omnipresent mesh-

work—and a lack of information on physiological function.

As recently as the early 1990s, one of the most prominent

neuroscience textbooks maintained that glial cells serve

mainly as bsupporting elements, providing firmness and

structure to the brain,Q and that they are bprobably not

essential for processing informationQ [145].In the last decade, we have learned much about glia.

Astrocytes, the most abundant cells in the CNS, secrete

many autocrine factors, as well as paracrine substances

which act on other types of glia, endothelial cells, and

neurons [10,24,47,179]. Astrocytes play a pivotal role in

the induction and maintenance of the blood–brain barrier

[24,123,139,160,248], and they communicate intracellular

Ca2+ signals bidirectionally with neurons [60,199,256].

They show robust responses to neurotransmitters such as

glutamate and norepinephrine [74,100], and signal back to

neurons by secreting neurotransmitters such as glutamate

themselves [47,219]. They also exhibit rapid electrical

activity in response to the firing of adjacent neurons

[195].

In an experimental model of long-term potentiation

(LTP), Wenzel et al. [280] observed that high frequency

stimulation of the perforant pathway caused astrocytes to

tightly wrap around the potentiated synapses, suggesting

that astrocytes may contribute to the cellular mechanisms of

learning. Anderson et al. [6] found that when rats were

taught new motor skills, the ratio of glia to Purkinje cells

increased, paralleling an increase in the number of synapses

per neuron.

Researchers have demonstrated that astrocytic Ca2+

signaling and calcium-induced transmitter release can

directly influence neuronal synaptogenesis and synaptic

transmission, providing further evidence of an astrocytic

contribution to neuronal plasticity and learning [9,15].

Araque et al. [10] pointed out that the ratio of glia to

neurons increases as one rises through the phylogenetic tree,

and the human brain has the highest ratio of glia to neurons

(N10:1).

2. Astrocyte responses to CNS disturbances

The astrocytic contribution to brain signaling is just

beginning to be investigated [10,30,47,60,87,124]. How-

ever, the astrocyte response to CNS injury and disease has

been the subject of innumberable investigations

[83,172,206]. Astrocytes proliferate during CNS develop-

ment, and then become quiescent. In regions adjacent to

CNS trauma or other pathology, astrocytes undergo

dramatic structural, biochemical, and functional trans-

formations collectively referred to as reactive gliosis.

These changes have long been recognized as some of

the earliest and most profound responses to CNS dis-

turbances [72,81].

Reactive gliosis is most commonly characterized by

changes in cell morphology and expression of GFAP, a

fibrillary intermediate filament that is specific for astrocytes

and rapidly upregulated in response to pathology

[72,76,77,81,182,194,207,236,281]. Numerous investiga-

tions have characterized GFAP expression in CNS injury

and disease since its discovery by Lawrence Eng and his

colleagues over thirty years ago (for review, see Ref. [81]).

The upregulation of GFAP is so stereotypical of the

astrocyte response to CNS pathology that the term breactiveastrocyteQ is often used simply to denote astrocytes staining

intensely for GFAP. Despite the homotypic upregulation of

GFAP, the time-course, extent, and specific details of the

astrocyte response can vary in different anatomical locations

and in specific disorders. A review by Fitch and Silver

documents several studies suggesting heterogeneity in the

glial reaction to different stimuli, especially regarding the

production of inhibitors of neuronal regeneration [92]. Other

components of the astrocyte response to CNS disturbances

include cytoskeletal remodeling, hypertrophy, proliferation,

and the secretion of growth factors, extracellular matrix, and

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508490

hormonal factors involved in recruiting inflammatory and

phagocytic cells, and further directing the response to injury

[77,164,203,206,281]. The magnitude and identity of these

responses can vary in certain circumstances, with the

balance determining the specific outcome. Therefore, using

the terms breactive astrocyteQ and bgliosisQ to encompass

these diverse responses can generate confusion. For the

purposes of this review, we will refrain from using the term

bgliosis,Q and instead refer to bastrocyte activationQ as any

changes in astrocyte functioning in response to CNS

pathology. We recognize that this encompasses many

diverse responses. Indeed, we hope to convey that the

dynamic nature of astrocytic mechanosensory mechanisms

may contribute to the generation of responses uniquely

tailored to the magnitude and type of insult.

Several investigations have focused on the stimuli

responsible for activating quiescent astrocytes. Since astro-

cytes become reactive at the periphery of insults, often

without any discernible direct connections to central

pathologic processes, researchers inferred that diffusible

factors must be important. Many different chemicals are

capable of activating quiescent astrocytes. These include

endothelin, interleukins, thrombin, neurotrophic factors, and

tumor necrosis factor alpha (TNFa) [92,176]. For a given

disturbance, multiple response pathways may interact,

accounting for some variability. Certain factors may be

specific for particular types of pathology, such as proteins

produced by tumor cells, or blood borne compounds that

can reach the brain through a compromised blood–brain

barrier. Other stimuli, such as interleukins and neurotrophic

factors, are released by neighboring neurons and microglia

[4,154,161,180].

Astrocytes are apparently able to modulate their function

in response to very specific environmental variables, and

thus are gaining notoriety for their phenotypic plasticity.

Primary astrocyte cultures exhibit remarkable heterogeneity

in properties such as receptor expression and Ca2+ signaling,

even among individual cells in the same culture [273,274].

For example, Shao et al. [251] showed that two astrocytes

derived from a single parent cell by a single mitosis can

exhibit different responses to neuroligands. Although the

specific details of the astrocyte response can vary, their

ability to respond to virtually any CNS disturbance with

both stereotyped changes (such as GFAP upregulation) and

an adaptable repertoire of other components suggests that

astrocytes may possess certain all-purpose sensors to

monitor changes in their local environment. This point

was recently acknowledged by Little and O’Callaghan, who

surmised that, b. . . the diversity of insults that engender

astrogliosis and the brain-wide nature of the astrocytic

response suggest that common injury factors serve as the

trigger of this cellular reaction.Q [169] The authors point outthat although numerous factors appear capable of influenc-

ing the course of gliosis, the bdamage factorsQ responsiblefor induction—which may be common to a variety of

disorders, remain uncharacterized.

3. Brain deformation and pathology

The name astro-cyte means bstar-shapedQ cell, referringto the numerous long processes that make multiple

connections with neurons, blood vessels, and other

astrocytes. Astrocytes form a three-dimensional meshwork

that extends throughout the brain—an ideal morphology

for sensing mechanical disturbances in the parenchyma. In

the early 1980s, prior to the demonstration of a mechano-

sensory apparatus in astrocytes, or even the ability of these

cells to communicate, Mathewson and Berry hypothesized

that,

b. . .astrocytes become reactive in response to architectural

disruption. . . the response is an attempt to stabilize the

morphological integrity of the damaged brain.Q [182]

The authors based this hypothesis on studies of cerebral

stab wounds in adult rats, from which they surmised that the

qualitative and temporal evolution of the astrocytic response

correlated with the distribution of mechanical stress result-

ing from the injury; i.e., the mechanical stresses appeared to

shape the brain’s response.

The symptoms of tumors, traumatic injuries, or other

CNS disturbances are often due to a mass effect. Indeed,

distortion of the surrounding brain is one of the basic

observations used in radiographic characterization of brain

lesions. Besides the direct deformation caused by trauma

or enlarging pathologic processes, the edema secondary to,

for example, cerebral infarction or injury creates signifi-

cant stress on the surrounding tissue. Additionally,

osmotic swelling of the astrocytes themselves, such as

demonstrated by Bullock et al. [41] within three hours of

head injury in patients undergoing surgery, generates

stress on the individual cells’ membranes and associated

structures.

Since nearly all brain pathology results in some degree of

deformation of the surrounding parenchyma, the ability of

astrocytes to respond to mechanical stress could provide a

general mechanism by which a variety of insults can be

managed by a single mechanism. The translation of

mechanical deformation into intracellular chemical signals

could account for the correlation between the spatio-

temporal characteristics of the response and the distribution

of stress, without requiring an extracellular diffusible

messenger. In this sense, mechanical deformation may

function as a primary stimulus that induces astrocytes to

alter their functioning.

4. Astrocyte mechanosensation

4.1. Stretch-induced Ca2+ waves

A large body of research has accumulated illustrating

that astrocytes can sense mechanical stress and translate it

into chemical messages, such as transmembrane ion fluxes

Fig. 1. Fluorescent imaging of mechanically induced calcium waves (from Ref. [214]). Note the shadow of the pipette tip in the top-left frame. Approximately

12 s have elapsed in these four frames. The illustration at the bottom shows deformation of a single cell in a confluent monolayer with the side of a pipette.

Approximate tip diameter=5 Am. Note the ~308 bend in the tip of the pipette, to prevent puncturing the cell.

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508 491

and intercellular calcium (Ca2+) waves. As shown in Fig.

1, lightly pressing on a single adult rat astrocyte in a

monolayer using a micropipette tip, elicits a rapid increase

in intracellular Ca2+ in the stimulated cell (visualized by

loading the cells with a Ca2+-sensitive fluorescent dye).

Following a short delay at the cell borders, the Ca2+ wave

reappears in neighboring cells and continues to propagate

through the monolayer, often spreading throughout the

entire coverslip in confluent cultures [214]. Without

further stimulation, intracellular Ca2+ slowly decreases

over 5–10 min, after which the response is repeatable.

Similar propagated Ca2+ signals were first demonstrated in

neonatal astrocytes by Cornell-Bell et al. [63] in response

to glutamate. These waves have been widely studied,

contributing to the demise of the opinion that astrocytes

serve a merely passive structural role in the CNS. Several

researchers have shown that propagated Ca2+ signaling

can be elicited in fetal/neonatal astrocyte cultures by

many different stimuli, including direct mechanical stress

[33,52,272], endothelins [32,57,78,272], hypoxia/ischemia

[75,85], ATP [25,57], glutamate [25,52,57,62,63,74,89,

109,151,272], histamine [25,130], GABA [25], norepi-

nephrine [25,74], serotonin [25], angiotensin II [25,97],

bradykinin [25,129], substance P [25], carbachol [48,272],

brain-derived neurotrophic factor [58], gp120 HIV-1

envelope glycoprotein [59], phenylephrine [74], lysophos-

pholipids [94], and kainate [109].

In 1965, Bullock and Horridge presented a definition of a

nervous system as follows,

ban organized constellation of cells specialized for the

repeated conduction of an excited state from receptor sites

or other neurons to effectors or to other neurons.Q [40]

The most primitive nervous systems respond to stim-

ulation by propagating a diffuse signal, exemplified by the

bnerve netsQ of coelenterates. The astrocyte network is not

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508492

unlike a primitive nervous system: it is composed of

interconnected cells that can respond to stimuli by

propagating a diffuse signal, in the form of an intercellular

Ca2+ wave that spreads from the point of stimulus to other

astrocytes and neurons. As suggested by M.J. Sanderson et

al., these Ca2+ signaling phenomena could serve to

organize cooperative cellular responses to local stimuli in

vivo [244].

The dependence and interdependence of a multitude of

diverse regulatory mechanisms on cell Ca2+ is difficult to

fathom. Cells seem to employ what Michael J. Berridge et

al., have referred to as a bcalcium toolkitQ in which enzymes,

ion channels, intracellular stores, inositol phosphates,

endogenous buffers, pumps, and exchangers combine to

produce an array of tightly controlled Ca2+ phenomena that

probably depend upon spatial localization of the compo-

nents [27,28]. In glial cells, intracellular Ca2+ affects cell

structure, gene expression, development, and proliferation

(for review, see Ref. [90]). Disturbances in glial Ca2+

accompany many neuropathological conditions, including

astrocytoma, trauma, stroke, and epilepsy [3,53,71,90,

105,121]. The astrocyte response to a specific disturbance

would be shaped by the unique milieu of local stimuli

(including mechanical stress), the distribution of their

sensors/receptors, the degree and mode of intercellular

communications, and the time course of the disturbances.

Specific downstream signaling events may require specific

Fig. 2. Acute puncture of a single cell in the absence of extracellular Ca2+. (A) Not

view (insert shows magnified and enhanced image of circled area). (B) The white

fluorescence, evident in panel A, is no longer present—probably due to leakag

surrounding the punctured astrocyte show an increase in Ca2+. (C) DIC image of

pipette tip has been pulled away, leaving a hole in the punctured cell (see magnifie

the video camera and used to help position the pipette tip.

dynamic changes in cytoplasmic Ca2+ levels. For example,

Berridge points out that stimulation of cell proliferation

appears to require sustained signaling. Since calcium stores

are finite, this suggests a necessity for Ca2+ influx from

extracellular sources [26,27]. If Ca2+ influx is enhanced,

then a new equilibrium between this influx, storage, and

efflux is established, resulting in a new basal intracellular

Ca2+ concentration.

When an individual astrocyte in a monolayer is deformed

by a micropipette, the initial increase in intracellular Ca2+ in

the directly stimulated cell involves both Ca2+ influx and

intracellular release [244,272]. There is conflicting evidence

concerning the dependence of intercellular wave initiation

on an influx of extracellular Ca2+. Charles et al. [52] found

that neonatal rat astrocyte cultures were capable of

propagating intercellular Ca2+ waves in the absence of

extracellular Ca2+. Only the response in the stimulated cell

was lost. However, in a similar culture system, Venance et

al. reported that initiation of the waves depended on an

influx of Ca2+ into the stimulated cell [272]. In our

experiments with adult rat astrocytes, when external Ca2+

was removed from the extracellular solution, the mechanical

stimulus did not elicit a Ca2+ wave [213]. If the pipette tip

punctured the cell, the cell became dark presumably by

leakage of dye. However, after a short delay, Ca2+ increased

in the cells surrounding the punctured astrocyte (see Fig. 2).

This propagated Ca2+ signal was limited to cells in the

e the shadow of the pipette tip overlying the cell in the middle of the field of

asterisk shows the location of the punctured cell. Note that the background

e of dye out of the punctured cell. Also note that only cells immediately

the field of view, showing the pipette tip in contact with the cell. (D) The

d insert). The black line beneath the cell in panels C and D is generated by

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508 493

immediate vicinity of the damaged cell and spread in a

concentric fashion suggesting involvement of a diffusible

extracellular messenger (such as ATP) released from the

damaged cell.

The conflicting results reported by different labs regard-

ing the requirement for external Ca2+ raise the possibility

that Ca2+ waves are easily influenced by details of the

culture system, but they do occur in situ as shown in

hippocampal slice preparations, isolated intact retinas, and

acutely isolated cells [51,70,118,201]. Despite some con-

flicting data in the literature, there are agreed upon

characteristics of mechanosensitive Ca2+ signaling in

astrocytes:

(1) In response to mechanical stimulation, intracellular

Ca2+ increases.

(2) This calcium signal is propagated from cell-to-cell in a

bregenerativeQ fashion, forming an inter-cellular wave.

(3) The wave involves both an influx component as well

as release from intracellular stores.

4.2. Mechanosensitive ion channels (MSCs)

One way for cells to produce a Ca2+ influx in response to

mechanical stress is via specialized mechanosensitive ion

channels (MSCs). Currently MSCs are the only known

primary cellular mechanical transducers. Originally discov-

ered in the early 1980’s [108], MSCs are found ubiqui-

tously, from Escherichia coli to mammals [192,237–239].

Mechanical forces govern the gating of these channels, just

as voltage determines gating of voltage-gated channels. The

most common type of MSC is the stretch-activated channel

(SAC), although there are also examples of stretch-

inactivated channels (SICs) that are active at rest and

closed by tension [211,212]. SACs have been demonstrated

in neonatal astrocytes [37,134], glioma cells [36,187] and

adult astrocytes derived from striatal gelfoam implants

[261]. The astrocyte SACs can be activated by direct

pressure applied to membrane patches isolated in micro-

pipettes, and by osmotic swelling (which stresses the cell

membrane). Most SACs in animal cells, including those

found in astrocytes, are selective for cations including Na+,

K+ and Ca2+. Interestingly, one of the SACs characterized in

neonatal astrocytes is curvature sensitive [37]. This channel

is selectively opened only by an inward deformation of the

cell membrane—an ideal situation for responding to

mechanical stress imparted by the mass effects of nearby

pathology.

Since both acutely isolated astrocytes and slice prepara-

tions exhibit voltage- and ligand-gated Ca2+ channels

[73,74,152], multiple Ca2+ influx pathways probably also

exist in vivo. The non-mechanical Ca2+ influx pathways

may be indirectly activated by stretch. For example, voltage-

activated Ca2+ channels can open when stretch-channels

cause depolarization. Such a mechanism has been proposed

by O’Connor and Kimelberg to account for the regulated

volume decrease (RVD) observed in astrocytes following

osmotic swelling [209]: hypotonic media causes water

influx that swells the cells. This stretches the membrane

and opens SACs, which cause depolarization and lead to

Ca2+ influx via voltage gated channels. The increased

intracellular Ca2+ would then activate Ca2+-dependent K+

and Cl� efflux to reduce the swelling.

4.3. Cytoskeletal remodeling

Besides SACs, there may be other stress-activated

signaling pathways, since the structural elements of the

cytoskeleton are themselves mechanically sensitive poly-

mers. For example, altered GFAP expression constitutes one

of the earliest astrocyte responses to CNS disturbances:

! Dietrich et al. [72] found increased levels of GFAP

mRNA within 30 min of traumatic brain injury in rats.

! Amaducci et al. [5] similarly demonstrated morphology

changes and intense GFAP staining in the periphery of

parietal cryogenic lesions in rat brains 30 min after the

injury, spreading throughout the white matter by 48 h.

! Mucke et al. [194] demonstrated that focal mechanical

brain injury results in induction of an astrocyte-specific

GFAP-lacZ transgene within one hour.

! Cancilla et al. [45] found that GFAP mRNA was

increased following cerebral freeze-injury in mice by 6

h, and the pattern of enhanced expression in the two

weeks post-injury followed the distribution of edema.

! Liu et al. [171] similarly detected increased GFAP

mRNA within 6-h in the peri-infarcted area induced by

middle cerebral artery occlusion in rats.

Several studies have demonstrated that GFAP plays a

role in maintaining normal astrocyte cell shape and

cytoskeletal structure [102,234–236]. In a review of the

potential roles of GFAP, James Rutka et al. pointed out that

this astrocyte-specific intermediate filament has long been

considered to function as a mechanical integrator of

cellular space [236]. The authors also highlighted studies

suggesting that GFAP plays a pivotal role in forming an

interconnected dynamic cell-signaling apparatus, in con-

junction with other components of the cytoskeleton,

extracellular matrix, and associated kinases and second

messengers.

In addition to the dramatic increase in GFAP expression

that characterizes astrocyte activation, there is a transient

upregulation of vimentin (another intermediate filament),

increased expression of bactin-anchoring proteinsQ such as

vinculin, talin, and paxillin (which help attach the actin

cytoskeleton to focal adhesions on the cell membrane), and

a remodeling of the actin cytoskeleton [2,127,144,164,

225,266]. Rapid cytoskeletal remodeling in response to

mechanical stress has been demonstrated in other cell types,

suggesting that this is prototypical. Pender and McCulloch,

for example, showed that the actin stress fibers of gingival

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508494

fibroblasts are disassembled within ten seconds of a stretch,

but recover completely within 1 min [221].

The actin cytoskeleton is also involved in astrocyte

signal transduction. Abd-el-Basset and Federoff demon-

strated that fetal astrocytes possess actin stress fibers

containing all of the elements traditionally considered to

make up a bcontractile unitQ—F-actin, myosin, tropomyo-

sin, and caldesmon, as well as the enzymes myosin light-

chain kinase (MLCK) and calmodulin [1]. Although

neonatal astrocytes exhibit extensive gap junctional cou-

pling shortly after subculture, Cotrina et al. [65] demon-

strated that they cannot propagate Ca2+ waves until this

actin cytoskeleton develops. The authors further demon-

strated that the radius of the propagated Ca2+ wave was

directly proportional to the fraction of cells with organized

stress fibers, and disruption of the actin cytoskeleton with

cytochalasin D or inhibition of myosin light chain kinase

attenuated the Ca2+ waves. Astrocyte stress-fibers in vitro

organize into parallel bundles that can span many cells

[1,65]. This arrangement allows the astrocyte network to

exert long range forces on the extracellular matrix and

produce coordinated changes in the structural scaffolding

of the surrounding brain.

4.4. Mechanosensation is dynamic

The stress-dependent reorganization of both intermediate

filaments (e.g. GFAP and vimentin) and the actin cytoske-

leton enable astrocytes to regulate the transduction of

mechanical forces in the brain, providing a time-dependence

to stress-induced stimulation. For example, a progressively

Fig. 3. Effects of Cyclic Stretch (1 Hz, 0–20%) on Inositol Triphosphate Productio

and exposed to cyclic deformation regimens using a Flexcell FX-3000 Strain Unit

bloadedQ into the membrane store of inositol (PIP2), and then is transferred to c

measurement of radioactivity in the cytoplasmic fraction reflects cumulative IP3 p

cytoplasmic IP3 concentration. Ca2+-free=1 h incubation with EGTA prior to stretc

bars=FSE.

increasing mechanical stress, such as a rapidly expanding

tumor mass, might continually stimulate a mechanosensory

element, whereas the response to an acute event, such as a

traumatic injury, may be attenuated as the mechanosensory

apparatus is remodeled.

The dynamic response of mechanical signaling systems

is exemplified by the intrinsic adaptation and inactivation of

the stretch-activated ion channels in different tissues

[114,205,261]. In what is presumably a more downstream

view of transduction, the mechanically induced turnover of

inositol phosphates in adult astrocyte cultures is also

dynamic [215]. Fig. 3 shows that a 1-Hz stretching regimen

applied to astrocytes increased the turnover of inositol

phosphates within 15 s, reaching a plateau within 2 min. In

other words, 2-min of periodic stretching was sufficient to

activate and then deactivate the mechanical signaling

machinery of the astrocytes. Remarkably, cessation of the

stretching was seen as a new stimulus, i.e. a change in the

mechanical environment, resulting in further IP3 production.

The correlation of this phenomenon to in situ stimuli is

unknown, but the normal mechanical environment for

astrocytes is pulsatile. Indeed, if a portion of the skull is

resected, the bbeatingQ of the brain is easily appreciated. As

with all sensory processes, there is a tonic and a phasic

response. Since the primary mechanotransducers are modu-

lated by the mechanics of their surroundings, any change in

the mechanical environment can serve as a stimulus. The

combination of cytoskeletal remodeling, dynamic second

messenger signaling, and the adaptation and inactivation of

the SACs themselves provide a complex multi-tiered time-

dependence to stress-activated signaling.

n (from Ref. [214].) Adult astrocyte cultures were grown on flexible plates

(Flexcell International, USA). Prior to stretching, tritiated myoinositol was

ytoplasmic IP3 when PLC-mediated turnover is stimulated. A subsequent

roduction. DPM=disintegrations per minute, which is proportional to mean

hing. N=3 wells for each data point (~9.6 cm2 growth area per well). Error

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508 495

5. Endothelins

Endothelin (ET) is a 21-amino-acid bicyclic peptide (see

Fig. 4). It was originally identified by Yanagisawa et al.

[286] in the conditioned medium of cultured endothelial

cells, by virtue of its actions as a potent and long-lasting

vasoconstrictor. Researchers have determined that there are

at least three peptides in the endothelin family—ET-1, ET-2,

and ET-3, and at least two main types of receptors—ETAand ETB [104,188]. The ET peptides and receptors are

expressed in a tissue-specific manner. Their properties have

been characterized in numerous reviews [104,106,126,

148,210,278]. ET-1 appears to be the predominant form in

humans, and for the remainder of this review, bET-1Q andbendothelinQ are used interchangeably. However, it is

important to note that researchers have demonstrated the

existence of both ET-1 and ET-3 in the human brain

[79,80,174].

5.1. Endothelin in astrocytes

Although not found in non-proliferating quiescent

astrocytes, immunohistochemical staining has demonstrated

that reactive astrocytes contain endothelin [142,173,174,

202,292–295]. ET is found in reactive astrocytes in the

periphery of a remarkably diverse collection of CNS

pathologies, including traumatic injuries [112,227], hema-

togenous metastases [292,293], plaques of Alzheimer’s

Disease [202,294,295], experimentally induced edema

[112,113], infarcts and lacunae [202,294], hereditary

multi-infarct disease [294], lesions of viral infections

[173,202] , and Binswanger’s encephalopathy [294]. Siren

et al. [255] reported a direct correlation between astrocyte

reactivity (quantified by GFAP levels and morphology

changes) and ET-1 expression in rat brains following

cryogenic neurotrauma. Koyama et al. [158] found that a

continuous infusion of a selective ETB receptor antagonists

into the cerebral ventricles of rats attenuated the increase in

GFAP- and vimentin-positive cells after a cerebral stab

wound. Tsang et al. [269] have recently shown that the

expression of ET-1 mRNA is increased following cerebral

hypoxia/ischemia.

When ET was first discovered in reactive astrocytes,

some researchers hypothesized that it had diffused to the

astrocytes from damaged blood vessels. Indeed, in rats

subjected to acute brain trauma by cold injury, Yamada et al.

[284] demonstrated that radiolabeled ET-1 injected into the

Fig. 4. Endothelin-1 molecule. Endothelin-1 is a 21 am

left cardiac ventricle was later detected in the brain at the

injury site. However, numerous studies have demonstrated

that activated astrocytes also produce, store, and secrete

endothelins themselves [79,80,174,202,269]. Astrocytes

(and glioma cells) possess endothelin receptors, and

endothelin binding can potentiate the production and

secretion of more endothelin in a positive feedback loop

[79,80,177,178].

ET receptors appear to be broadly distributed in

astrocytes. For example, Venance et al. [273] found that

all striatal astrocytes in rat brain express binding sites for

ET-1, whereas a1-adrenergic and muscarinic binding sites

are more heterogeneously distributed. Although astrocyte

ET-receptors are distributed throughout the brain, the

relative density of ETA and ETB receptors varies in

different regions, depends on the state of differentiation,

and is altered in response to certain diseases and trauma

[98,104,111,112,196,218,227]. ETA and ETB receptors are

distinguished by their affinities for the ET-isopeptides

[210]. ETA receptors have a higher affinity for ET-1 and

ET-2 than ET-3, with Kd’s of ~20–60 pM vs. ~6.5 nM,

respectively. ETB receptors bind all three isopeptides with

equivalent affinities (Kd ~15 pM). Several researchers have

characterized ET binding as birreversibleQ—owing to their

inability to wash off bound radiolabeled ET from its

receptors [56,181,282,283]. This high affinity binding is

further demonstrated by comparing the short half-life of

ET-1 injected into the blood stream (a few minutes) with

the resultant long-lasting vasoconstriction (hours)

[253,286]. Evidence exists for additional atypical ET-

receptors, including an astrocyte-specific receptor that

may have multiple binding sites [141]. There is also ET-

binding in a human hybrid neuroblastoma/glioma cell line

that has a higher affinity than ETA or ETB receptors [7].

In addition to acting as a vasoconstrictor, numerous

studies have revealed that ET stimulates the proliferation

of particular cell types, including astrocytes. ET-1

immediately transforms differentiated, non-proliferating,

astrocyte cultures into proliferating cells, with a saturating

effect at nanomolar concentrations [49,111–113,147,165,

174,258,263]. Hama et al. [112] demonstrated that

astrocytes change from a quiescent to an immature reactive

state immediately after experimentally induced acute brain

injury in vivo, and the level of endothelin is significantly

increased at the site of injury after only 1 day. ET is also

mitogenic for numerous other cell types, including glioma

cells [13,68,249,250], brain capillary endothelial cells

ino acid, bicyclic peptide, weighing 2491.9 kD.

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508496

[275], glomerular mesangial cells [155,247,254,290],

smooth muscle cells [14,175,252] (especially vascular

smooth muscle [14,132,138,156]), normal prostate epithe-

lium [133], prostate cancer cells [200], and ovarian

carcinoma cells [16].

Another component of the brain’s reaction to pathology

is infiltration of the site of injury by leukocytes [19], and

implantation of activated leukocytes caused behavioral

improvement in a rat model of Parkinson’s Disease [82].

Although not well studied in the brain, endothelin has pro-

inflammatory actions in the lungs, intestinal mucosa, blood

vessels, and cutaneous sites, which include stimulating

leukocyte recruitment [241–243], enhancement of leukocyte

adhesion and infiltration [35,107], macrophage activation

[186,189], and enhanced edema, microvascular leakage, and

nociception [101,107,222]. ET also stimulates the produc-

tion of other inflammatory cytokines including TNFa,

interleukins, and interferon gamma [91,230,257]. These

inflammatory actions are best characterized in the lungs,

where ET appears to play a major role in the pathogenesis of

asthma and immune-complex-mediated acute lung injury

[22,50,88,91,101,257]. Therefore, it is possible that ET

contributes to the pathophysiology of leukocyte recruitment

observed in the brain as well.

5.2. Endothelin regulation

It is generally recognized that there is an intimate

relationship between mechanical forces and endothelin.

Indeed, ET was initially discovered because of its most

dfamousT role as a potent and long-lasting vasoconstrictor.

Conversely, mechanical stretch is a major stimulus for ET-1

production and release. Alterations in hemodynamic shear

stress, such as might result from increased blood pressure or

heart rate, can modify ET-1 production and secretion by the

endothelium [190,191,277,291]. Endothelin then acts on

the vascular smooth muscle to regulate vessel tone.

Mechanical stress has been directly implicated in increasing

endothelin levels in several pathological conditions, includ-

ing pulmonary hypertension [270], cardiac hypertrophy

[135,136,285], atherosclerosis [120], neointimal hyperpla-

sia following coronary angioplasty [167,184], and excess

flow through the adrenal gland [44]. Additionally, gross

alterations of the mechanical environment characterize

numerous other pathologies associated with elevated

endothelin, where the role of direct cell deformation has

yet to be investigated. Some examples include persistent

pulmonary hypertension of the neonate [146,229], primary

glaucoma (i.e. raised intraocular pressure) [110,143,

208,223], mesangial proliferative nephritis [290], solid

tumor growth (i.e. mass effect) in gliomas, ovarian

carcinomas, and advanced prostate cancer [13,16,17,116,

200,260], acute and chronic renal failure [148,228],

numerous obstructive, hypertensive, and inflammatory

pulmonary diseases [101,122,226], neurotrauma [21,

246,255], bladder outlet obstruction [149,150], and pre-

eclampsia [42,84,276]. The pathogenesis of many of these

disease processes involves both the vasoconstricting and the

mitogenic actions of endothelin.

Although the expression of the ET isopeptides, convert-

ing enzymes, receptors, and other components of the

astrocytic endothelin system have been identified, consid-

erably less is understood about the regulation of this system.

As characterized in an editorial by Hannelore Ehrenreich,

bthe players within the astrocytic endothelin (ET) system are

known, but the rules of the game have been obscure. . .Q[78]. Compared to the work on astrocytes, regulation of the

ET system in other cells has been examined more

extensively. Besides stretch, several non-mechanical stimuli

can enhance ET expression, including thrombin, TGF-h,angiotensin II, arginine vasopressin, calcium ionophores,

and a host of other growth factors and cytokines (for

reviews, see Refs. [104,181,268]). Regardless of the

stimulus, alterations in cell Ca2+ play a central role in

regulating the endothelin system in all cell types

[11,38,39,61,181,191,268,287]. Abnormal levels of ET are

usually caused by increased transcription and/or secretion

(frequently upregulated in parallel), which are controlled by

intracellular Ca2+ [38,39,268]. This regulation has histor-

ically been considered to rely on a Ca2+-dependent

induction of mRNA transcription, involving the proto-

oncogene products c-fos, c-myc, and c-jun, followed by

secretion via a bconstitutiveQ pathway without appreciable

intracellular storage [104,181,286,287]. The unique gene

structure of ET-1, which contains several bacute phase

reactionQ motifs and nuclear binding factors, allows for the

rapid induction of gene transcription [131]. Intracellular

Ca2+ homeostasis has also been implicated in regulating the

unstimulated basal secretion of ET-1 by endothelial cells

[38,39], and in stimulating secretion by activating myosin

light chain kinase (MLCK), which facilitates the transfer of

ET-1 to the cell surface [153].

Even in early experiments in the vasculature, it was

apparent that certain stimuli elicited increased plasma ET-1

concentrations much faster than would be expected from de

novo synthesis. For example, Fyhrquist et al. [96] showed

that rapidly submerging a subject’s forearm into ice cold

water induced an approximately seven-fold increase in

plasma ET-1 in the arm within 2 min. Researchers have

demonstrated a second bregulatedQ secretion pathway in

endothelial cells, involving storage of mature ET-1 in

secretory vesicles and endothelial-cell-specific storage

granules called Wiebel-Palade bodies, and subsequent rapid

release induced by chemical or mechanical stimuli that

trigger a Ca2+ influx [119,231–233]. Similarly, Nakamura et

al. [197] found ET-1 co-localized with vasopressin and

oxytocin in neurosecretory granules of the rat posterior

pituitary. Thus, Ca2+ signaling controls both the rapid and

transient secretion of preformed stores of ET via the

bregulatedQ pathway, and the somewhat slower regulation

of ET-mRNA transcription, followed by secretion via the

bconstitutiveQ pathway.

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508 497

In experiments with endothelial cells, Chen et al. [55]

used a bmagnetic twisting stimulatorQ to directly apply

mechanical stress to integrin receptors on endothelial cells

via ligand-coated ferromagnetic beads bound to the cell

surface. This resulted in dramatically enhanced expression

levels of ET-1 mRNA. The authors further showed that this

ET-1 induction could be modulated by cytoskeletal dis-

ruption, and was abolished by blocking stretch-activated ion

channels with Gd3+, or by removing extracellular Ca2+. This

work demonstrates:

(1) The importance of cytoskeletal components in modu-

lating cellular mechanosensitivity.

(2) The critical role of SACs as proximal mechanotrans-

ducers.

(3) The dependence of this mechanical signaling mecha-

nism on an influx of Ca2+, presumably via SACs.

ET-1 exerts its mitogenic effects in most cell types,

including astrocytes and glioma cells, by regulating intra-

cellular Ca2+ homeostasis [128,137,177,178,263,264],

potentially explaining how endothelin potentiates its own

production and secretion. Although the specifics of this

signaling cascade may vary among cell types, the mitogenic

actions of ET appear to converge on activation of the

mitogen-activated protein kinase (MAPK) pathway through

Ca2+, the induction of phospholipase C (PLC), and protein

kinase C (PKC) [104]. PLC mediates the turnover and

intracellular release of inositol triphosphate (IP3), which

diffuses through gap junctions and causes release of

intracellular Ca2+ stores in neighboring cells. This produces

a propagated intercellular Ca2+ wave. ET-1 is an extremely

potent stimulus of astrocytic IP3 production. In neonatal

astrocyte cultures, ET-1 administration caused an upregula-

tion of PLC (and subsequent increase in cytoplasmic IP3)

greater than that induced by glutamate, carbachol, or

methoxamine [272]. Additionally, Laurent Venance, Chris-

tian Giaume et al. [272,273] found that Ca2+ waves elicited

by either local application of ET-1 to a single cell or

mechanical stimulation elicited robust Ca2+ waves that

propagated throughout the astrocyte network whereas other

stimuli produced considerably smaller responses.

The capacity for ET to enhance its own expression

necessitates feedback inhibition. The existence of such

regulatory mechanisms is demonstrated by experiments

showing that under certain conditions, increased concen-

trations of ET-1 or ET-3 can inhibit gap junctional

permeability and the propagation of intercellular Ca2+

waves in neonatal astrocytes [32,265]. Additionally, Corder

et al. found that prolonged exposure to calcium ionophores

actually inhibited ET secretion by bovine aortic endothelial

cells [61].

The dramatic cytoskeletal remodeling that characterizes

the astrocyte response to pathology may favor specific

regulatory pathways by altering connections between

cellular components, or varying the relative densities of

individual cytoskeletal elements. For example, Ozawa et al.

[218] found that disruption of microtubules attenuated both

the expression of ETB receptor mRNA and the number of

ET binding sites on neonatal rat astrocytes, while micro-

filament disrupting agents had no effect. The potent self-

stimulating capacity of the ET system, combined with its

apparently multifactorial feedback regulation, suggests that

prolonged alterations or disruptions at any of several points

along these signaling cascades could result in dramatically

abnormal expression levels.

The relationship between mechanical stretch and ET

production in other cell types, combined with the observa-

tion that ET-1-positive reactive astrocytes appear in the

mechanically deformed periphery of CNS pathology, led us

to hypothesize that mechanical stress may regulate ET in

astrocytes. In our experiments with adult astrocytes grown

on flexible culture dishes, a cyclic stretching regimen

caused a dramatic increase in ET-1 production and secretion

[213,214]. Since virtually all brain pathology results in

mechanical deformation of the surrounding parenchyma,

and ET induces the proliferation of astrocytes (and glioma

cells), the possibility arises that mechanical induction of the

ET system represents a general pathway for activating or

augmenting astroglial proliferation.

Alterations in the astrocyte production of ET may affect

other cells as well. Although astrocytes are considerably

more sensitive than neurons to the mitogenic actions of ET-

1, the sensitivity of both cell types exceeds that of most

peripheral tissues [262]. Additionally, several studies have

implicated ET-1 in aspects of central autonomic control (for

review, see Kuwaki et al. [162]), and the cerebral

vasculature is particularly sensitive to the vasoconstricting

actions of ET-1 [12,95,240,267]. ET concentrations as low

as 1–100 pM affect the proliferation of astrocytes, glioma

cells and neurons, induce the contraction of cerebral and

basilar arteries, alter extracellular glutamate and dopamine

levels, and prevent cytochalasin B-induced astrocyte mor-

phology changes (see Table 1).

6. Potential therapeutic targets

6.1. ET-receptor antagonists as treatments for brain injury

Currently, experimental treatments for diseases related to

the overproduction of ET primarily focus on blocking the

effects of the peptide using receptor antagonists. Another

developing class of drugs blocks ET-converting enzymes

(ECEs), preventing the cleavage of a precursor form into

mature endothelin [140]. Endothelin-receptor antagonists

inhibit the endothelin-induced proliferation of glioma cells

in vitro [249,250]. They also reduce the degree of edema,

infarct volume, and neurological deficits in rats following an

ischemic event [20,21,183,220]. Similarly, they attenuate

neurological deficits and edema following traumatic closed-

head injury [21], control cerebral vasospasm [64], and

Table 1

ET-1 Effects in the brain at concentrationsV100 pM

Cell/Tissue Type ET-1 Conc. Effect Reference

Rat astrocytes in organotypic

cerebellar slices

EC50=8F5 pM Induced c-fos expression (correlates with mitogenicity). Sullivan and Morton,

1996 [262]

Rat neonatal cerebellar

astrocytes in mixed

astro/neuron cultures

(neuron enriched)

10–100 pM Threshold concentration for eliciting increases in intracellular

calcium. Maximal response elicited at ~10 nM ET-1.

Morton and Davenport,

1992 [193]

Primary rat astrocytes EC50=50 pM Inhibited cytochalasin B-induced stellation. Koyama and Baba,

1994 [157]

100 pM Stimulated mitogen-activated protein kinase (MAPK) activity. Kasuya et al., 1994 [147]

100 pM–100 nM Dose-dependent inhibition of glutamate uptake. Leonova et al., 2001

[166]

EC50=100 pM Stimulated proliferation (i.e. increased DNA synthesis

measured by [3H]thymidine incorporation).

MacCumber et al., 1990

[174]

100 pM Stimulated proliferation (i.e. increased DNA synthesis

measured by [3H]thymidine incorporation).

Stanimirovic et al., 1995

[258]

Rat neurons (granule cells) in

organotypic cerebellar slices

10–1000 pM

(EC50=57F9 pM)

Induced c-fos expression (correlates with mitogenicity). Sullivan and Morton,

1996 [262]

Human cerebral cortex Kd=25F6 pM [125I]ET-1 binding studies in membrane preps. Fernandezdurango et al.,

1994 [86]

Ventral striatum of

anaesthetized rats (in vivo)

10 pmol injection 8 AM increase in extracellular dopamine concentration

within 5 min of injection.

Webber et al., 1998

[279]

C6 glioma cells EC50=50 pM Stimulated proliferation (i.e. increased DNA synthesis

measured by [3H] thymidine incorporation).

MacCumber et al.,

1990 [174]

Canine basilar arteries

(in vivo)

10 pmol intracisternal

injection

Decrease in artery diameter (measured by angiography);

remained statistically significant 3 days after injection.

Asano et al., 1993 [12]

Canine cerebral arteries

(endothelium denuded)

10 pM–100 nM Dose-dependent stimulation of contraction. Contractile effects

between 10 and 300 pM were significantly greater than for

mesenteric arteries.

Tanoi et al., 1991 [267]

Porcine cerebral arteries

(ring segments)

100–300 pM Threshold concentration for ET-1 induced contractions. Fukuda et al., 1991 [95]

Examples of mitogenic and vasoconstricting actions induced by administration of endothelin.

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508498

reduce both the size of lesions resulting from cold-injury

[103] and the degree of acute neuronal damage following

mild cortical trauma in rats [246]. These drugs are also

being investigated as possible treatments for a variety of

other cerebrovascular and neoplastic pathologies [46,116,

117].

Although ET-antagonists have shown promise in treating

certain conditions, they do not address the underlying

pathophysiology. Since ET plays numerous roles in normal

cell physiology throughout the body, blocking the receptors

can lead to multiple side effects. The high sensitivity of the

cerebral vasculature to ET [240], coupled with the delicate

regulation of blood flow in the brain and the role of ET in

maintaining basal vascular tone [125,126], suggests that the

potent vasoactive properties of ET-related peptides may

limit their usefulness in treating CNS pathology. By

characterizing the upstream cell-signaling pathways

involved in regulating ET production, treatments may be

designed to target the causes of ET overproduction, rather

than using receptor antagonists to block the effects.

6.2. Inhibitors of stretch-activated ion channels (SACs)

Since mechanical stress is a well characterized stimulus

for the ET system throughout the body (and this appears to

involve a stretch-induced Ca2+ influx), and pathological

processes cause mechanical perturbations, SACs represent a

logical target for inhibiting this system. The bgold-standardQtechnique for implicating an individual type of ion channel

or class of channels in a particular process is to determine

whether a specific inhibitor alters the results. Although

patch-clamping techniques have enabled the identification

and kinetic analysis of SACs in numerous cell types, the

lack of specific pharmacological agents has hindered

elucidation of their physiological roles. The lanthanide

gadolinium (Gd3+) is an effective blocker for many SACs,

and therefore is often used to implicate these channels in

cellular phenomena [115,288]. For example, Gd3+ inhibits

the stretch-induced Ca2+ influx and proliferation of fetal rat

lung cells, while Ca2+ channel blockers such as verapamil,

nifedipine, and Ni2+ have no effect [170]. However Gd3+

also blocks voltage dependent Ca2+-channels [31,163], and

is not a suitable reagent for experiments in most culture

media, as it precipitates in the presence of proteins,

phosphates, or bicarbonate [34,43,238]. Certain amino-

glycoside antibiotics (e.g. streptomycin) are also capable

of blocking SACs as well as other cation channels. These

positively charged molecules appear to function as blockers

by bpluggingQ the negatively charged pore of the channel

[115,159].

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508 499

Vaz et al. [271] showed that several of these somewhat

non-specific SAC inhibitors, including gadolinium, amilor-

ide, and gentamicin, inhibit edema in vivo following head

trauma. The effects were statistically significant, even when

the SAC blockers were administered 15–30 min after the

trauma. Although it is not possible to distinguish effects on

individual cell types in their model, the authors hypothe-

sized that the reduced edema was due to inhibition of SACs

activated by endothelial cell swelling. Recently, this same

group has demonstrated that both gadolinium and amiloride

prevent alterations in brain mitochondrial function (strongly

affected by cell Ca2+) in their trauma model [245]. These

results are, at best, suggestive and need to be repeated under

more controlled conditions, since gadolinium was adminis-

tered i.v. and would be bound by proteins and small anions

[34,43,238].

In the mid-1990s, venom from the Grammastola

spatulata spider was shown to be capable of blocking

SACs in several systems, without affecting Ca2+ or other

voltage dependent channels [55,198,204]. More recently, we

have isolated and characterized a peptide from this venom

as the first specific blocker of stretch-activated cation

currents [261]. This 34-amino-acid peptide, named GsMtx-

4, belongs to the inhibitor cystine knot (ICK) family—in

which an embedded ring is formed by two disulfide bonds

and their connecting backbone, and penetrated by a third

disulfide bond (see Fig. 5). The disulfide bonds stabilize the

native conformation, and because of the stability of the

structure, these peptides are appealing candidates for drug

design [69,224].

In adult rat astrocytes, GsMtx-4 completely blocks the

stretch-activated channels, while exhibiting no effect on

voltage-sensitive currents [261]. GsMtx-4 also inhibits

swelling-activated currents in the adult astrocytes [261].

Preliminary data additionally shows that the stretch-induced

secretion of ET-1 is inhibited by GsMtx-4 [215]. GsMtx-4

Fig. 5. Ribbon Structure of GsMtx-4, illustrating binhibitor cystine knotQ(ICK) motif. A ring is formed by two disulfide bonds and their connecting

backbone (blue), and penetrated by a third disulfide bond (yellow). The

inset is an illustration of the bcystine knotQ structure from Craik et al. [69].

did not alter the basal ET-1 secretion in the absence of

stretch, illustrating the specific nature of its actions.

Streptomycin, which inhibits stretch-activated currents in

several systems, also attenuated this response, as did

reducing extracellular Ca2+. These data suggest that

mechanically induced ET-1 production by astrocytes is

dependent on an influx of Ca2+ [213]—similar to the

situation in endothelial cells [54].

The cDNA sequence encoding the GsMtx-4 peptide has

been identified, and both a synthetic and a recombinantly

cloned form have been successfully created [216]. Addi-

tionally, the solution structure has been determined using

two-dimensional NMR spectroscopy [217]. The synthetic

form is now available from The Peptide Institute, (Osaka,

Japan). Mutagenesis studies on the recombinant peptide to

determine which amino acids are critical for binding and

activity are underway. It is our hope that these studies may

result in an entirely new class of drugs for the treatment of

CNS pathologies.

7. Mechanical deformation and endothelin in

astrocyte-derived tumors

Gliomas of astrocytic origin are the most common type

of human brain tumors and are almost invariably fatal. For

the most malignant variety, glioblastoma multiforme, there

has been no significant improvement in life-expectancy

during the past 15–20 years. Li et al. [168] suggest that the

relative frequency of these tumors reflects the sensitivity of

glial cells to proliferative stimuli. As the authors point out,

the vast number of proliferatively quiescent astrocytes in the

CNS, combined with their ability to re-enter the cell cycle

upon stimulation necessitates tight control over cell pro-

liferation. This apparently fails in neoplasia. Glioma cell

proliferation is particularly sensitive to changes in Ca2+

[23], and it has been suggested that this is due to

overexpression of a Ca2+-dependent isoform of PKC

[18,29]. PKC activity directly correlates with the prolifer-

ation rates of astrocytes and gliomas [66,67,289]. Addition-

ally, both human and rat glioma cell lines have high levels

of PKC compared to non-malignant astrocytes [67].

The mass effect of enlarging gliomas is so characteristic

of their behavior that distortion of the surrounding

parenchyma provides the basis for their radiographic

detection, and is used to gauge malignant behavior. Like

non-malignant astrocytes, glioma cells possess SACs and

generate intracellular Ca2+ signals in response to mechanical

stimulation [37,53,93,187]. Several studies have demon-

strated a correlation between increasing astrocyte malig-

nancy and decreasing GFAP expression (for review, see

Ref. [236]). Since GFAP has long been considered to play a

role in stabilizing the cytoskeleton and astrocyte cell shape,

it appears that there may be a correlation between the loss of

normal structural integrity (a situation that would alter

mechanosensation), and the development of neoplasia.

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508500

Additionally, if astrocytoma cell lines are transfected with

GFAP, there is an inverse relationship between the resulting

mRNA expression and cell proliferation in vitro [234].

Like non-malignant reactive astrocytes, glioma cells

produce and secrete endothelin which is mitogenic for

glioma cells in vitro [13,68,116,249,250] and in vivo

[116,259,260]. Endothelin antagonists can inhibit glioma

cell proliferation in vitro [249,250]. Stiles et al. [260]

demonstrated a strong correlation between ET-immunor-

eactivity in surgical tissue sections from human gliomas and

tumor malignancy. Further supporting the role of ET in

neoplasia, Stiles et al. showed that if a human glioma line

that is a low endothelin producer is transfected with a gene

to increase ET production, the malignancy of the tumors

resulting from xenografts into the brains of immunodeficient

rats is increased. Studies have also demonstrated significant

differences in the degree of ET-receptor expression, receptor

classes, capacities for ET clearance, and sensitivities to ET-

1-induced Ca2+/IP3 signals and mitogenesis, between

glioma strains of different malignancies and normal

astrocytes [7,8,174,282,283].

The expression of ET-1 in human astrocytomas correlates

with the vascularity of the tumors [260]. Because ET is a

potent mitogen for both capillary endothelium and vascular

smooth muscle, it has been implicated in angiogenesis

[99,260,294,296], the process whereby tumors develop their

own vasculature. Angiogenesis is necessary in order for a

solid tumor to grow larger than a few millimeters. In

glioblastoma multiforme, the most malignant variety of

astrocyte-derived gliomas, the vascular proliferation is so

striking that it is used as a diagnostic criterion to distinguish

the tumors from their less malignant counterparts [185].

Glioblastomas are essentially inoperable and invariably

fatal, primarily because of their diffusely infiltrative nature.

However, partial resection, termed debulking, often pro-

longs survival [3]. The most obvious benefit of this

procedure is a reduction of the tumor cell load; however

debulking would also lessen the pressure tending to deform

the surrounding brain.

The less malignant low-grade astrocytomas grow much

more slowly and are relatively avascular, until transforming

into more malignant tumors. A hypothetical explanation for

the behavior of these tumors might be that during their

initial slow growth, the tissue is being gradually deformed.

At some point, a threshold of mechanical deformation is

reached, sharply increasing endothelin production. The

endothelin then stimulates angiogenesis, and the resulting

vascular proliferation enables the more rapid, malignant

growth of the tumor. This would produce greater mechanical

strain, perpetuating the cycle.

Fig. 6. Regulation of the Endothelin System in Astrocytes. See text for

references and discussion.

8. Conclusions

Hypotheses that historically relegated astrocytes to a

structural and supportive role were inspired by the reticular

meshwork formed by these stellate cells. Rather than

precluding an active role in cell-signaling, this arrangement

places the astrocytes in an ideal situation to monitor,

measure, and integrate the effects of mechanical stress.

Astrocytes possess complex mechanosensory machinery,

which includes

(1) dynamically adapting cytoskeletal components that

regulate force transduction,

(2) stretch-activated ion channels, which directly sense the

mechanical stress and translate it into ion fluxes,

(3) mechanically induced second-messenger signaling

phenomena, such as intercellular Ca2+ and IP3 waves,

and

(4) the stretch-induced production and secretion of endo-

thelin, a potent astrocyte mitogen (with additional

affects on neurons, brain capillary endothelium, and

glioma cells).

Fig. 6 summarizes the pathways involved in regulating

endothelin in astrocytes. Mechanically induced Ca2+ and IP3waves are well-established astrocyte phenomena. Both ET-1

and increased intracellular Ca2+ enhance astrocyte prolifer-

ation [49,90,112,147,165,174,258,263]. Additionally, we

have shown that mechanical deformation directly stimulates

the production and secretion of ET-1 by adult astrocytes

[214]. Since virtually all brain pathology deforms the

surrounding parenchyma, astrocyte mechanosensation can

facilitate the observed stereotypical reactive response to a

diversity of insults. The existence of mechanosensory

phenomena does not preclude contributions of chemical

and electrical signaling cascades in astrocyte function.

Additionally, it is likely that the other cells of the brain

L.W. Ostrow, F. Sachs / Brain Research Reviews 48 (2005) 488–508 501

possess stress-signaling mechanisms themselves. However,

the unique morphology and dynamic stress-sensing and

modulating abilities of astrocytes make them ideal for

monitoring and responding to mechanical disturbances.

CNS pathology necessarily causes mechanical stress on

the surrounding tissue. Astrocytes possess mechanisms to

sense this stress, translate it into chemical signals, and

regulate its transduction in an adaptable time-dependent

manner. The upregulation of the astrocyte ET system

observed in a wide range of pathologies, coupled with its

potent effects on these cells, suggests a prominent role in

this response. Therefore, we believe that mechanical

induction of the astrocytic endothelin system represents a

largely unexplored, and potentially significant target for

therapeutic intervention.

Acknowledgments

We would like to thank JST and NIH for support.

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