an increasing number of individual transport proteins and
their genes, their functions and interactions being explored
and their defects becoming known. This article is an attempt
to illuminate this widespread field. It could not properly pay
tribute to those many investigators responsible for these
numerous new discoveries, who lead us to a better understanding of liver transport functions.
References
Ceramides as Key Players in Cellular Stress Response
Josef Pfeilschifter and Andrea Huwiler
The recent discovery of sphingolipid-derived second messengers that regulate fundamental cell
responses such as cell growth and apoptosis has provided insight into the way cells sense and
respond to stressful stimuli. This will help the understanding of the pathogenesis of stress-related
diseases and eventually offer novel therapeutic approaches.
L
iving cells are often exposed to dramatic changes in environmental conditions and adapt to these changes by expression of inducible enzyme systems that provide an appropriate
response to the environmental signals. However, the physiological systems activated by stress stimuli can not only protect
but may also damage the cell. Obviously, regulation of these
seemingly contradictory functions must operate on multiple
pathways to ensure an adequate homeostasis. Researchers
need to find out how these multiple signaling pathways are
coordinated. This review focuses on ceramide, a lipid second
messenger generated by sphingomyelin hydrolysis that was
recently identified as a key player in stress signaling in mammalian cells (3, 12, 14, 15).
J. Pfeilschifter and A. Huwiler are in Zentrum der Pharmakologie, Klinikum
der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590
Frankfurt am Main, Germany.
0886-1714/99 5.00 © 2000 Int. Union Physiol. Sci./Am.Physiol. Soc.
Sphingolipid-mediated signal transduction
Sphingolipids are derivatives of long-chain bases and display
a great structural diversity and complexity. They have a headgroup at position 1 of the ceramide backbone, such as phosphorylcholine to form sphingomyelin and carbohydrates to
form glycosphingolipids. Sphingomyelin is located predominantly in the outer leaflet of the plasma membrane. Biosynthesis of sphingolipids (Fig. 1) starts with the condensation of
serine and palmitoyl-CoA to yield 3-ketosphinganine, which is
reduced further to sphinganine and converted to dihydroceramide by the enzyme ceramide synthase. In the next step,
headgroups are added to form sphingomyelin and glycosphingolipids. This occurs mainly in the Golgi apparatus and, in
some cases, in the plasma membrane.
Degradation of sphingolipids begins with the removal of
headgroups. In the case of sphingomyelin, phosphorylcholine
News Physiol. Sci. • Volume 15 • February 2000
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1. Boyer, J. L., J. Graf, and P. J. Meier. Hepatic transport systems regulating pHi,
cell volume, and bile secretion. Annu. Rev. Physiol. 54: 415–438, 1992.
2. Crawford, A. R., A. J. Smith, V. C. Hatch, R. P. J. Oude Elferink, P. Borst,
and J. M. Crawford. Hepatic secretion of phospholipid vesicles in the
mouse critically depends on mdr2 or MDR3 p-glycoprotein expression.
J. Clin. Invest. 100: 2562–2567, 1997.
3. Fitz, J. G., and J. A. Cohn. Biology and pathophysiology of CFTR and other
Cl– channels in biliary epithelial cells. In: Biliary and Pancreatic Ductal
Epithelia, edited by A. E. Sirica and D. S. Longnecke. New York: Marcel
Dekker, 1997, p. 107–125.
4. Gerloff, T., B. Stieger, B. Hagenbuch, J. Madon, L. Landmann, J. Roth, A. F.
Hofmann, and P. J. Meier. The sister of P-glycoprotein represents the
canalicular bile salt export pump of mammalian liver. J. Biol. Chem. 273:
10046–10050, 1998.
5. Gorboulev, V., J. C. Ulzheimer, A. Akhoundova, I. Ulzheimer-Teuber, U.
Karbach, S. Quester, C. Baumann, F. Lang, A. E. Busch, and H. Koepsell.
Cloning and characterization of two human polyspecific organic cation
transporters. DNA Cell Biol. 16: 871–881, 1997.
6. Hagenbuch, B., and P. J. Meier. Sinusoidal (basolateral) bile salt uptake
systems of hepatocytes. Semin. Liver Dis. 16: 129–136, 1996.
7. Hofmann, A. F. Bile acids: the good, the bad, the ugly. News Physiol. Sci.
14: 24–29, 1999.
8. Keppler, D., and I. M. Arias. Transport across the hepatocyte canalicular
membrane. FASEB J. 11: 15–18, 1997.
9. Kullak-Ublick, G.-A., B. Hagenbuch, B. Stieger, C. D. Schteingart, A. F.
Hofmann, A. W. Wolkoff, and P. J. Meier. Molecular and functional characterization of an organic anion transporting polypeptide (OATP) cloned
from human liver. Gastroenterology 109: 1274–1282, 1995.
10. Lee, T. K., L. Li., and N. Ballatori. Hepatic glutathione and glutathione
S-conjugate transport mechanism. Yale J. Biol. Med. 70: 287–300, 1997.
11. Müller, M., and P. L. M. Jansen. The secretory function of the liver: new
aspects of hepatobiliary transport. J. Hepatol. 28: 344–354, 1998.
12. Oude Elferink, R. P. J., D. K. Meijer, F. Kuipers, P. L. Jansen, A. K. Groen,
and G. M. Groothuis. Hepatobiliary secretion of organic compounds:
molecular mechanisms of membrane transport. Biochim. Biophys. Acta
1241: 215–268, 1995.
13. Paulusma, C. C., M. Kool, P. J. Bosma, G. L. Scheffer, F. ter-Borg, R. J.
Scheper, G. N. Tytgat, P. Borst, F. Baas, and R. P. J. Oude Elferink. A mutation in the human canalicular multispecific organic anion transporter gene
causes the Dubin-Johnson syndrome. Hepatology 25: 1539–1542, 1997.
14. Trauner, M., P. J. Meier, and J. L. Boyer. Molecular pathogenesis of
cholestasis. New Engl. J. Med. 339: 1217–1227, 1998.
15. Zsembery, A., C. Spirli, A. Granato, N. F. LaRusso, L. Okolicsanyi, G.
Crepaldi, and M. Strazzabosco. Purinergic regulation of acid/base transport
in human and rat biliary epithelial cell lines. Hepatology 28: 914–920, 1998.
is released by acidic, neutral, and Mg2+-dependent sphingomyelinases to yield ceramide. Indeed, ceramide is a central metabolite in sphingolipid catabolism and is degraded to
fatty acid and free long-chain bases by neutral or acidic
ceramidases. The sphingosine thus formed can be reacylated
subsequently to ceramide, phosphorylated to yield sphingosine-1-phosphate, or methylated. Several of these intermediates (ceramide, sphingosine, and sphingosine-1-phosphate)
have potential roles as second messengers, with ceramide
being the best characterized (14).
A number of cytokines, growth factors, and other environmental stress stimuli employ the sphingomyelin-signaling pathway to generate ceramide (Table 1). The mechanism coupling
the receptors of the ligands or the physical stimuli to activation
of sphingomyelinases are poorly understood. It is assumed that
physical stresses act directly on the membrane and, by poorly
defined processes, trigger activation of an acidic sphingomyelinase and subsequent signaling through the stress-activated protein kinase (SAPK) cascade. The best-characterized
membrane receptor known to activate the sphingomyelin pathway is the 55-kDa tumor necrosis factor (TNF) receptor that
binds TNF-a and is able to deliver rapid signals to a neutral and
an acidic sphingomyelinase. This receptor possesses two distinct cytoplasmic domains, one of which links a novel adaptor
protein, designated FAN (factor associated with neutral sphingomyelinase activation) to activation of a neutral sphingomyelinase and ceramide production. The other domain,
which is identical to the death domain of the 55-kDa TNF
receptor, couples to the proapoptotic adapter proteins TRADD
(TNF receptor-associated death domain) and FADD (Fas/Apo-1associated death domain) and is suggested to link to the acidic
sphingomyelinase (1).
Irrespective of the stimulus used and the receptor system
involved, it is quite obvious that several mechanisms exist that
12
News Physiol. Sci. • Volume 15 • February 2000
initiate ceramide production and that each cell type may display a specific pattern of activated sphingolipid pathways.
Identification of ceramide targets
With respect to immediate targets of ceramide, suggestions
for several ceramide-activated enzymes have been forwarded.
These include a 97-kDa proline-directed serine/threonine protein kinase, which recently has been suggested to be identical
to the kinase suppressor of Ras (KSR) (12), a ceramide-activated protein phosphatase of the okadaic acid-sensitive PP2A
subgroup of protein phosphatases (3), diverse protein kinase C
isoenzymes (8, 10), and the protein kinase c-Raf (6). Addition
of ceramide to intact cells or the pharmacological modulation
of endogenous levels of ceramide alters the activity of members of the mitogen-activated protein kinase (MAPK) cascades, the classic extracellular signal-regulated kinases (ERK)
1 and 2, and the more recently discovered SAPKs of the
SAPK/c-Jun NH2-terminal kinase (JNK) subfamily or the
p38/reactivating kinase (RK) subfamily. These parallel signaling pathways regulate such fundamental aspects of cell function as metabolism, secretion, and gene expression. The
archetypal signaling cascade includes ERK1 and ERK2 and
responds primarily to mitogenic stimulation via the small G protein Ras. In contrast, the SAPK and p38 pathways respond to
cellular stresses such as inflammatory mediators, poisons,
heat, and high-energy radiation. However, there are cell typespecific patterns of activation of the different MAPK cascades
by a given ligand, which determine the cell-specific response
to this stimulus. Despite intensive efforts in recent years, identification of the molecular targets of ceramide action has
proved difficult and so far indirect. It is clear, however, that
ceramide delivers signals into one or several cascades of the
MAPK families in a cell type-specific manner to regulate cell
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FIGURE 1. Biosynthetic and catabolic pathways of sphingolipids. DAG, 1,2-diacylglycerol; PC, phosphatidylcholine.
TABLE 1.
Stimuli known to stimulate sphingolipid turnover and ceramide production
Inducers of
Apoptosis
TNF-a
Fas ligand
Dexamethasone
Nitric oxide
Inducers of
Differentiation
Vitamin D3
TNF-a
Nerve growth factor
Shear stress
Retinoic acid
Progesterone
Damaging
Agents
Ionizing radiation
UV light
Heat shock
Oxidative stress
Daunorubicin
Vincristine
Inflammatory
Cytokines
IL-1a
IL-1b
TNF-a
Interferon-g
TNF-a, tumor necrosis factor-a; IL, interleukin.
cally binds to c-Raf and stimulates its kinase activity, thus
establishing ceramide as a lipid second messenger that acts as
a major direct activator of c-Raf in IL-1b- and TNF-a-triggered
signal propagation.
Interestingly, PKC-a and PKC-d isoforms were identified as
direct targets of ceramide. No binding of ceramide to PKC-ε
and PKC-z, the other isoenzymes of PKC present in mesangial
cells (5), could be detected. Binding of ceramide to PKC-a is
accompanied by an increase in kinase activity in vitro. In vivo
activation of PKC-a by ceramide was monitored by delayed
translocation of the isoform from the cytosol to the membrane
fraction of mesangial cells. Unexpected was the finding that
PKC-z, in contrast to other reports (10), was not labeled with
[125I]TID-ceramide in mesangial cells (6). This again suggests
that ceramide may interact with potential targets in a cell typespecific manner (8).
An important question that immediately arises is concerned
with the molecular mechanism of ceramide binding to its
direct targets identified so far, i.e., PKC-a, PKC-d, and c-Raf (6,
8). Is there a specific binding motif for ceramide that is common in these molecules? The conserved C1 and C2 domains in
the regulatory part of PKC isoenzymes are candidates for lipidinteracting motifs. The C2 part present in the conventional
cPKCs contains a Ca2+-phospholipid-binding domain (CaLB
domain) that is responsible for interaction with phospholipids.
The C1 part present in all PKC isoenzymes contains tandem
repeats of cysteine-rich motifs, a so-called zinc butterfly, that is
thought to be responsible for binding of DAG and phorbol
esters (4). A list of potential further ceramide targets containing
either a CaLB domain or zinc butterfly or both is shown in
Table 2. A homologous cysteine-rich motif is present in c-Raf.
Although these lipid-binding motifs share certain common
characteristics, they are not functionally equivalent, which
may explain why only PKC-a and PKC-d, but not PKC-ε or
PKC-z, are able to directly bind ceramide (7).
PKC as a switching-off device of sphingolipid signaling
Nishizuka and his colleagues first suggested that DAG generated from hormone-induced phosphoinositide hydrolysis
provides the physiological activator of PKC (11). PKC and
cytosolic free Ca2+ were demonstrated to act independently or
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responses such as proliferation, apoptosis, or gene expression
and subsequent mediator synthesis.
A novel approach to identify downstream targets of ceramide
is the use of a radioiodinated, photoaffinity-labeling analog of
ceramide with high 125I-specific radioactivity (>2000 Ci/mmol)
designated [125I]3-trifluoromethyl-3-(m-iodophenyl)diazirine
(TID)-ceramide. On irradiation with light at ~350 nm, a photolabile diazirine moiety is rapidly photolyzed to generate a
carbene capable of reacting with the full range of functional groups occurring in biomolecules (2). This technique
enables the cell physiologist to take snapshots of even very
transient interactions between cell molecules and the photocrosslinker used. This tool has been successfully employed
in identifying c-Raf as a ceramide-binding partner in renal
mesangial cells (6), a cell type orchestrating inflammatory
processes in the renal glomerulus and thus predestinated to
process stress signals (13).
Ceramide not only bound to c-Raf but also increased protein
kinase c-Raf activity. The signal was further processed along
the MAPK-ERK kinase (MEK)/ERK cascade and caused an
increased activity of ERK1 and ERK2 that could be inhibited by
a synthetic inhibitor of MEK. Importantly, the physiological
agonist interleukin (IL)-1b was found to use this signaling pathway to activate c-Raf in mesangial cells (6).
The serine/threonine kinase c-Raf is a well-studied signaling
device that is responsible for phosphorylation and activation
of MEK in the classic MAPK cascade. The mechanism of
activation of c-Raf has been studied extensively, and it is
now clear that this is a multistep event. In the first step, c-Raf
translocates to the plasma membrane and associates with RasGTP. This association with Ras-GTP is not sufficient for c-Raf
activation but is required for its recruitment to the plasma
membrane. In the next step, c-Raf is activated by an unknown
mechanism that may comprise a tyrosine and/or serine/threonine phosphorylation of c-Raf or the interaction with another
membrane cofactor such as a lipid. The c-Raf NH2 terminus
contains a highly conserved region (CR-1) that encompasses a
zinc-finger motif analogous to the lipid-binding domain of
protein kinase C (PKC), and it is tempting to speculate that
c-Raf is activated by the binding of a lipid second messenger
in a manner similar to the mechanism of activation of PKC by
1,2-diacylglycerol (DAG). It is obvious that ceramide specifi-
TABLE 2. Potential ceramide targets containing a cysteine-rich
lipid-binding domain (zinc butterfly) or a Ca2+-phospholipid
binding domain (CaLB domain)
Proteins with a zinc butterfly
A-Raf, B-Raf, c-Raf
PKC-a, -b, -g, -d, -ε, -h, -u, -m, -z, -i (l)
Myosin heavy-chain kinase
N-chimaerin
Vav
DAG-kinase
Unc-13
KSR
Proteins with
a CaLB domain
PKC, protein kinase C; DAG, 1,2-diacylglycerol; PLC,
phospholipase C; cPLA2, cytosolic phospholipase A2; PI3K,
phosphatidylinositol 3-kinase; RACKS, receptor for activated
C kinase; KSR, kinase suppressor of Ras; GAP, GTPase-activating protein.
Perspectives
synergistically to initiate a myriad of receptor-controlled cell
responses. An equally important function of PKC is the immediate negative feedback control of agonist-induced phosphoinositide turnover to prevent overshot of the system, thus establishing
PKC as a bidirectional regulator of cellular functions (11). PKC-a
isoenzyme was shown to be responsible for feedback regulation
of hormone-stimulated inositol lipid hydrolysis in glomerular
mesangial and endothelial cells (5, 7), which may be of importance in the mechanisms underlying homologous or heterolo-
With ceramide, the list of lipid second messengers has
adopted a novel and prominent member derived from sphingomyelin turnover. By introducing photocrosslinkers to identify ceramide targets, it will be possible to trace the molecular
chain that possibly constitutes one of the major signaling pathways by which messages carried by stress stimuli are transmitted from the cell membrane to the cell interior, where the
appropriate response required to deal successfully with environmental changes is initiated.
FIGURE 2. Feedback regulation of phosphoinositide and sphingomyelin pathways by protein kinase C (PKC). PLC, phospholipase C; SMase, sphingomyelinase.
14
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PKC-a, -b, -g
PLC-b, -g, -d
p120rasGAP
cPLA2
Synaptotagmin
Unc-13
Rabphilin-3A
RACKS
PI3K-C2a, -C2b
PI3K-68D/cpk
gous desensitization (5). In this context, it is worth mentioning
that ceramide also triggers PKC-a-mediated feedback inhibition
of inositol lipid signaling (8). Since ceramide has a stimulatory
effect on PKC-a activity (see above), it was tempting to speculate
that this activation might be physiologically relevant and be followed by inhibition of inositol phosphate formation in mesangial
cells. Indeed, it was observed that preincubation of cells for up
to 2 h with ceramide time-dependently blocked extracellular
nucleotide-evoked inositol trisphosphate generation (8).
Moreover, in analogy to feedback regulation of phosphoinositide turnover, it has been reported that IL-1b-induced
ceramide formation is inhibited by stimulation of PKC and that
this effect is reversed by specific PKC inhibitors or by downregulation of PKC isoenzymes in mesangial cells (9). From these
data, it is tempting to speculate that endogenous ceramide,
formed in mesangial cells after cytokine stimulation, activates
PKC-a, thereby establishing a negative feedback loop that
switches off ceramide production (Fig. 2). One can envisage
that cross-talk between the phosphoinositide- and sphingomyelin-signaling pathways engaging PKC-a provides an elegant and comprehensive mechanism of autoregulation.
We gratefully acknowledge the support of the Deutsche Forschungsgemeinschaft (SFB 553), the Wilhelm Sander-Stiftung, and the Commission of the
European Communities (Biomed 2, PL 90979).
References
Extracellular Surface Charges in Voltage-Gated
Ion Channels
Michael Madeja
A large number of charged amino acids are in the extracellularly located parts of the voltage-gated
ion channels. Recent findings suggest that these surface charges contribute to the channel functions
in the sensing of voltage, the binding of substances, and the sensing of H+ concentration.
V
oltage-gated ion channels play important roles in the functioning of several cell types. Ion channels have thus
become one of the major objects in physiological research.
Although the function of ion channels has been under investigation for a few decades, only in recent years has insight into
the structural properties of voltage-gated ion channels been
added (cf. Ref. 11). Although the complete three-dimensional
structure of the voltage-gated ion channel is still unknown, the
structure of the pore region has been determined by X-ray
crystallography of a bacterial potassium channel (2), and
topological models have been developed on the basis of
hydrophobicity profiles and mutagenesis experiments (Fig. 1).
The channel—or at least the central part carrying the pore and
the voltage sensor—is composed of four basic structural elements that are termed subunits, domains, or repeats (Fig. 1A).
Each element consists of an amino acid chain that is thought
to have six putative a-helical membrane-spanning segments,
termed S1 to S6 (Fig. 1, B and C). The amino and carboxy ter-
M. Madeja is in the Institute for Physiology, University of Münster, RobertKoch-Str. 27a D-48149 Münster, Germany.
0886-1714/99 5.00 © 2000 Int. Union Physiol. Sci./Am.Physiol. Soc.
mini of the amino acid chain face the cell interior. The linkers
connecting the segments S1 with S2, S3 with S4, and, in part,
S5 with S6 are assumed to be located extracellularly and to
form, at least for the most part, the extracellular surface of the
voltage-gated ion channels.
For several regions of the ion channel molecule the functional role has been predicted. Experimental studies have
especially focused on the central part of the region between
segments S5 and S6, which forms a major part of the ion conducting pathway (p region or pore; Ref. 2), whereas the S4 segment has been shown to comprise at least part of the voltage
sensor (cf. Ref. 12). Although a large number of studies have
dealt with the structure-function relation of these regions, comparably little attention has been paid to the structure-function
relation of the extracelluarly located linkers of the channel molecule. The charged amino acids of these regions are of particular interest because they contribute to an extracellular surface
charge, which might affect the process of voltage gating and
binding of extracellularly applied substances and protons.
In this article, the possible function of the charged amino
acids in the extracellular linkers is summarized and discussed.
Thus attention is focused on the glutamate and aspartate
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