Thapsigargin And Agonist Mediated Release Of Calcium Biology Essay

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Thapsigargin has been used prodigiously as a tool to elucidate the mechanisms of store-operated calcium entry. Critically evaluate how useful thapsigargin has been in furthering our understanding of the regulation of calcium fluxes in mammalian cells.

The agonist-mediated release of Ca2+ from endoplasmic reticulum (ER) or its specialized subcompartments is one of the central themes of chemical signalling in cells. This mode of signalling is dependent on an active sequestration of Ca2+ within the stores, a task accomplished by a class of transporters termed sarco-endoplasmic reticulum Ca2+-ATPases (SERCAs). Thapsigargin is the most widely used SERCA inhibitor. Thapsigargin inhibits the skeletal muscle sarcoplasmic reticulum Ca2'-ATPase with a dissociation constant lower than 1 nM [43. The formation of the thapsigargin enzyme complex is stoichiometric (1: 1) and inhibits the Ca2'-dependent hydrolysis activity and the formation of the phosphorylated intermediate. Calcium binding to the Ca"-ATPase is also affected by thapsigargin: it has also been reported that only a single calcium ion would bind to the Ca*'-ATPase after inhibition by thapsigargin and that this remaining bound Ca" would not be located on the high-affinity binding external sites, but binds to the enzyme with a relatively lower affinity.

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It was first shown to increase free cytosolic Ca2+ in platelets in 1985, and by 1994, an average of more than one report daily involving thapsigargin was being registered by Medline.

Thapsigargin was isolated from the Mediterranean plant Thapsia garganica L. (Linnaeus), and its structure was elucidated by a combination of chemical, spectroscopic and X-ray-crystallographic methods. It belongs to a group of related, naturally occurring 6,12- guaianolides with a 1b-disposed hydrogen and a 7b-disposed hydroxy group, found in several species belonging to the genus Thapsia. The high lipid solubility of these compounds accounts for their excellent penetration of biological membranes.

Potency of thapsigargin towards SERCAs

The SERCA family comprises products of at least three

genes. The occurrence of SERCA1 is restricted to the

fast-twitch skeletal muscle fibres. SERCA2a is expressed

predominantly in slow-twitch and heart muscle while

SERCA2b appears to be expressed universally. SERCA3

is mainly found in the intestine and lymphatic tissues,

but has also been described in blood platelets, cerebellum

and PC12 cells. In general, the thapsigargin-mediated

inhibition of Ca2+ pumping to intracellular stores

was found to occur in the nanomolar range. However, a

considerable variation in the effective concentrations has

been reported. It is therefore

important to ask to what extent a significant variation

in the thapsigargin affinity between Ca2+-ATPase subtypes,

as opposed to other experimental variables, could

account for these data.

The very high affinity of thapsigargin for SERCA1

results in a stoichiometric binding, allowing the titration

of the enzyme based either on a change in intrinsic fluorescence

or activity. If a similar situation applies to

other SERCAs, the observed differences in the apparent

inhibitory potency will be influenced by the amounts of

ATPase per cell (or per mg microsomal preparation), as

well as the amounts of total cell (or microsomal) protein

in the assay. This has been demonstrated in work on

platelets, fibroblasts and adrenal chromaffin cells.

Thus, ATPase concentrations need to be kept constant

between various cell systems when looking for possible

multiple components of Ca2+-ATPase inhibition.

Such an approach has been attempted in blood

platelets. In platelets, 10 nM and 100-200 nM thapsigargin

induced a partial and a complete emptying of Ca2+

stores, respectively. This suggestion of a molecular

diversity of platelet SERCAs was supported by the

finding that maximally effective concentrations of the

inhibitor 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ) were able to mimic the Ca2+-releasing effect of high, but

not of low, concentrations of thapsigargin. In another

platelet study, a similar dissociation between the apparent

affinities of thapsigargin and tBHQ was described,

with the latter agent found to be a more potent inhibitor

of Ca2+ uptake to an inositol(1,4,5)-trisphosphate

[Ins(1,4,5)P3]-releasable than to an Ins(1,4,5)P3-insensitive

Ca2+ pool. The reverse conclusion applied to thapsigargin.

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Since thrombocytes have been shown to contain

SERCA2b and SERCA3 as well as a putative

new SERCA subtype, the general interest of these

results is in assessing the usefulness of thapsigargin as a

tool for pharmacological differentiation between SERCA

isozymes. However, it should be stressed that in these

papers, the thapsigargin data alone would not be enough

to sustain the claim of pump diversity. Use of tBHQ, a

pump-subtype-specific antibody, partial tryptic digestion

and ATPase autophosphorylation were all crucial

for the notion of the platelet multi-SERCA system.

Therefore, although a minor variation in the thapsigargin

inhibitory potency among the currently identified

SERCAs may exist, it is not sufficient to produce a truly

discriminating, biphasic inhibition profile in assays emulating

physiological conditions, such as ATP-hydrolysis

or an ATP-dependent Ca2+ uptake.

Do other SERCAs with truly low affinity for thapsigargin

exist? About 20% of 45Ca2+ loading to the stores in

permeabilized DC-3F fibroblasts was inhibited by thapsigargin

concentrations over four orders of magnitude

greater than the subnanomolar range required for inhibition

of the major uptake component. The presence of

thapsigargin in a long-term culture promoted a selection

of cells in which such thapsigargin-resistant Ca2+-

ATPases became dominant. Similar thapsigarginin sensitive

Ca2+-ATPases may exist in other cell types.

The exact relationship between the structural and

functional properties of this type of pump and the

known SERCA isoforms presents an intriguing question.

The fact that thapsigargin-insensitive pumps may be

specifically associated with Ca2+ stores of distinct properties

[e.g. devoid of Ins(1,4,5)P3 receptors] highlights

the usefulness of thapsigargin as a dissecting tool for

studies of molecular and functional aspects of Ca2+

physiology in cells.

Specificity of thapsigargin towards SERCAs

The apparent specificity of thapsigargin towards

SERCAs is remarkable. Nevertheless, there is evidence

suggesting that caution should be exercised, especially in

the micromolar concentration range. For example, thapsigargin

in this range appeared to inhibit the store-operated

Ca2+ entry35. While 5 nM thapsigargin or 500 nM tBHQ

depleted the Ins(1,4,5)P3-sensitive stores in GH3 pituitary

cells, each of these agents at 5 mM was shown to block the

L-type Ca2+ channels. Such observations indicate that

when working with whole cells or other complex systems,

one would be well advised to test the specificity of

any effects presumed to reflect the inhibition of SERCAs

by thapsigargin in micromolar concentrations. Alternative

blockers tBHQ and cyclopiazonic acid are readily

available for such purpose. While more difficult to

obtain, sterical isomers of thapsigargin displaying as

much as a 3000-fold lower inhibitory potency towards

SERCA activity offer a strong testing criterion.

Thapsigargin and Ca2+ release from intracellular

stores

Release of Ca2+ from intracellular stores by thapsigargin

accounts for most of its popularity in the literature.

However, it achieves this effect indirectly, as opposed to

ionophores such as calcimycin or ionomycin. Inhibition

of SERCAs by thapsigargin prevents the pumps from

counterbalancing the passive Ca2+ leak from the stores to

the cytosol. Thus, the Ca2+-releasing action of thapsigargin

depends on two factors simultaneously: the presence

of thapsigargin-sensitive pumps and an appreciable

rate of Ca2+ leak (and thus of Ca2+ turnover).

As discussed by Pozzan and colleagues, leak rates may

vary among systems. If, under particular experimental conditions, this passive Ca2+ leak is slow on the time

scale of the experiment, even a complete pump arrest by

thapsigargin will cause no Ca2+ discharge. Alternatively,

the effect of a Ca2+-releasing agent [such as Ins(1,4,5)P3

or caffeine], whether applied directly or invoked by an

agonist, will not be prevented under these conditions

by a previous thapsigargin exposure. Claims of the

presence of thapsigargin-insensitive Ca2+ pumps have

occasionally been based on such results, but similar

future postulates should be supported by additional

tests such as those described below.

(1) In intact cells, the apparent inability of thapsigargin

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to discharge (a portion of) the intracellular agonistsensitive

Ca2+ pool should also be tested in the presence

of a low concentration of ionomycin, which by itself

induces only very modest Ca2+ efflux29. This will assess

whether the lack of thapsigargin sensitivity, and not of a

sufficient Ca2+ turnover, underlies the missing response.

(2) If an agonist-mediated Ca2+ discharge does not

appear to be prevented by a previous exposure to thapsigargin,

any effect due to the slow Ca2+ turnover can be

eliminated by a demonstration that such failing inhibition

also occurs during at least one additional release

cycle, after the stores have been reloaded in the presence

of thapsigargin.

(3) Provided that experimental control over the ATP

or Ca2+ supply to the pumps is possible (such as in permeabilized

cells or microsomes), thapsigargin may be

added before the onset of Ca2+ uptake. This type of

approach has been used to ascertain the dependence of

both caffeine- and Ins(1,4,5)P3-sensitive Ca2+ stores on

thapsigargin-sensitive pumps38, as well as the presence

of a truly thapsigargin-insensitive component of Ca2+

uptake18,27.

Thapsigargin and a role of SERCAs in

generation of Ca2+ signals

One important question concerning SERCAs is

whether their role in Ca2+-store-dependent signalling

extends beyond refilling the stores after an agonist-mediated

Ca2+ discharge. Is a cell's total ER pump capacity

regulated in a moment-to-moment manner? Would such

regulation affect the generation of Ca2+ signals and the

cellular response to an agonist? In the heart, the activity

of SERCA2a is regulated by an interaction with phospholamban,

a membrane protein whose phosphorylation

by cAMP-dependent protein kinase and Ca2+/

calmodulin-dependent protein kinase (CaM kinase)

increases pump affinity for Ca2+ (Ref. 39). Physiologically,

this provides a catecholamine-mediated mechanism

of systole shortening and augmenting heart contractility.

By contrast, no clear-cut regulatory mechanism

for any of the other SERCAs is known today. However,

putative regulation of non-muscle SERCAs has been

suggested40. A CaM kinase phosphorylation site, shown

in vitro to accept phosphate in SERCA2a (Ref. 41), is

shared by SERCA2b (Ref. 2).

Studies using thapsigargin have demonstrated the

functional relevance of such a search for SERCA regulation.

In patch-clamped pancreas acinar cells, 500pM

thapsigargin was used to partially inhibit SERCAs without

any increase in the resting cytosolic Ca2+ concentration42. This partial inhibition was sufficient to change some

characteristics of the Ca2+ signal: the duration of individual

Ins(2,4,5)P3-induced Ca2+ spikes as well as the spike

frequency and the sensitivity to Ins(2,4,5)P3 were

increased. A model was proposed to explain these

results in which the decreased rate of Ca2+ removal by

the pumps led to an increased occupancy of cytosolic

Ca2+-binding capacity. The remaining and diminished

cytosolic Ca2+ buffering would then enable a smaller

amount of Ca2+ entering the cytosol to activate the

Ins(1,4,5)P3 receptor and initiate spiking.

In the above example, thapsigargin has shown that

SERCAs in non-muscle tissues may be a meaningful

regulation target. Furthermore, it has demonstrated

some of the possible consequences that an agonist-mediated

control of SERCA activity, if indeed present, might

have for generation of Ca2+ signals. For such studies in

whole cells, in which many system components need to

remain intact (including channels, receptors and cytosolic

proteins, as well as membrane and organelle

integrity), the high penetrability and potency of thapsigargin

make it particularly suitable, minimizing the

likelihood of nonspecific effects.

One important aspect of Ca2+-signal generation

involves the role of store-operated Ca2+ entry. This mode

of Ca2+ entry (also known as the capacitative entry)

through channels in the plasma membrane is activated

by the emptying of Ca2+ stores, and appears to play an

important role in numerous cellular responses ranging

from volume regulation to proliferation43. Work in this

area has provided another good illustration of the specificity

of thapsigargin-mediated SERCA inhibition. When

activation of plasma membrane Ca2+ permeability secondary

to inhibition of Ca2+-store pumps was studied in

rat thymocytes, it was found that, although maximally

effective concentrations of thapsigargin, tBHQ or cyclopiazonic

acid resulted in a depletion of Ca2+ stores to a

similar degree, the two latter compounds also caused a

partial blockade of the external Ca2+ entry35. By contrast,

concentrations of thapsigargin tenfold larger (about

300 nM) than those necessary for maximal store depletion

did not interfere with the store-operated activation of

Ca2+ influx at the plasma membrane. However, inhibition

became apparent when concentrations of thapsigargin

in the micromolar range were tested, defining the

limits of its specificity in this system.

Thapsigargin and a role for Ca2+ stores in cell

growth

The very tight binding of thapsigargin to the currently

known SERCAs results in inhibition being practically

irreversible, persisting after dilution44 or removal of the

excess inhibitor. This property of thapsigargin has been

used to study the process of pump synthesis and Ca2+-

pool reconstitution in smooth-muscle cells following

an 18-hour application and subsequent removal of

thapsigargin45. A close correlation had previously been

observed between the activity of the pumps in keeping

the Ins(1,4,5)P3-sensitive Ca2+ stores filled and the ability

of the cells to maintain protein and DNA synthesis and

full cell-cycle progression20. Following the removal of

excess thapsigargin, the 'old-pump'-arrested state was

retained while permitting the cells to recover; thus, the

time window and medium composition essential for the

induction of 'new-pump' synthesis, the reappearance of

Ca2+ pools, and the resumption of DNA synthesis and

cell proliferation could be determined45. Hence, in this

case thapsigargin has promoted an inquiry into one of

the least illuminated and yet important aspects of the

role of Ca2+ stores, that of securing proper conditions for

cell division.

A noteworthy example of a cell-biological correlate to

the previously mentioned distinction between the mechanism

of Ca2+-releasing action of thapsigargin and that of

Ca2+ ionophores has been provided by these studies.

While a complete pump arrest in the smooth-muscle

cells by thapsigargin was shown to stop cell proliferation

and caused the Ins(1,4,5)P3-sensitive Ca2+ pool to remain

empty for seven days, the overall cell morphology and

viability were not affected. By contrast, even low concentrations

of calcimycin caused the death of almost all

cells within 24 hours20.

Thapsigargin: a drug lead?

The long-standing interest in the potential medical

benefits of Thapsia garganica is attested by writings of

Theophrastos (c. 372-287 BC) and by its use in traditional

Arabian and European medicine for rheumatic pains, up

to its listing in the French Pharmacopoeia of 1937. Modern

work using purified thapsigargin uncovered its

tumour-promoting action on mouse skin46. Following an

initial rise, the fraction of mice developing tumours

unexpectedly dropped before a much slower rise ensued

after long-term thapsigargin exposure46. More recently,

it has become clear that application of thapsigargin is

associated with activation of programmed cell death in a

number of cell types24,25,47. This apoptotic aspect of thapsigargin

action might account for the transient decline of

tumours in the mice assay mentioned above. Importantly,

it may also be seen as an opportunity to develop

a thapsigargin-derived anti-cancer drug. A foremost

problem in any such application will be the selective and

efficient targeting of the drug to the malignant cells. As

one possible strategy, an inactive prodrug might be constructed,

designed to liberate an active thapsigargin

analogue at a location restricted by the presence of

hydrolyzing enzymes specifically associated with the

tumour.

Concluding remarks

Close to 20 years following its isolation6, thapsigargin

has come of age as a potent and remarkably specific

tool for studying Ca2+ stores and their SERCAs. Provided

that attention is paid to issues such as stoichiometry and

irreversibility of binding, the indirect mode of Ca2+-store

depletion, and a possibility - never to be discounted - of nonspecific effects, thapsigargin may be considered a

highly useful addition to a pharmacologist's toolbox,

and one possibly harbouring a therapeutic potential.

TOPOGRAPHY

Ca2.-ATPase belongs to the family of P-type ion

pumps, all of which use ATP to generate ion gradients

across a wide variety of cellular membranes.1

The best-studied pumps appear to use the same,

basic reaction cycle, the hallmark of which is transient

phosphorylation of an aspartate residue in

the catalytic site and cycling between two main

conformations, dubbed E1 and E2.2 More speci®-

cally, these conformational changes serve to couple

the energy of the aspartyl phosphate with translocation

of ions across the membrane. Recent evidence

suggests that these conformational changes

involve dramatic rearrangement of the cytoplasmic

domains with surprisingly small changes amongst

the ten transmembrane helices. Originally, these

structural dynamics were revealed at 8 AÊ by cryoelectron

microscopy (cryoEM) by comparing structures

of Ca2.-ATPase3 and H.-ATPase from

Neurospora,4 which were crystallized in different

conformations.5 A more recent, X-ray structure of

Ca2.-ATPase revealed the atomic architecture of

the E1 conformation and comparison with the

cryoEM map indicated large movements of the

cytoplasmic domains in response to Ca2. binding.6

For cryoEM of Ca2.-ATPase, the particular conformational

state required for crystallization was

stabilized by adding the inhibitor thapsigargin

(TG). This potent inhibitor is highly speci®c for the Ca2. pumps from sarcoplasmic and endoplasmic

reticula, known as SERCA, and was discovered as

the major source of skin irritation from root

extracts of Thapsia garganica, which were originally

used for treatment of muscle and joint in¯ammation.

7 Today, TG is widely used by cell biologists

wishing to empty the internal Ca2. stores in

eukaryotic cells, a result of total inhibition of the

SERCA pumps.

TG inhibits Ca2.-ATPase by binding to the E2

conformation and by preventing further cycling of

the enzyme.8,9 Normally, the E2 reaction intermediate

is converted to the E1 intermediate by the binding

of cytoplasmic Ca2. to their high-af®nity sites,

which in turn activates the ATP-binding site to

produce the phosphoenzyme intermediate (E1

_ P).

In the presence of Ca2., TG does not inhibit the

enzyme immediately, but allows a single cycle of

phosphorylation and ion transport, indicating that

TG can bind to only the E2 state. This interaction is

of very high af®nity (Kd < 1 nM) and full inhibition

is obtained with a 1:1 molar stoichiometry in what

has been termed a dead-end complex. Tubular

crystals require the E2 state, as indicated by their

requirement for low Ca2. concentrations,10 as well

as decavanadate,11 which mediates intermolecular

contacts as well as intramolecular interactions

between the three cytoplasmic domains.12 TG

stabilizes these crystals,9 presumably by making

them insensitive to Ca2. and by irreversibly trapping

all Ca2 .-ATPase molecules in a conformation

that is favorable for crystallization.

For our earlier cryoEM structure of Ca2.-

ATPase,3 we used the dansylated form of TG

(DTG),13 in order to stabilize the tubular crystals,

and with the hope that its larger size would

increase our chances of locating its binding site.

We have now solved several other structures in the

presence and absence of TG at 8-10 AÊ resolution

and calculated difference maps. These maps reveal

a consistent, high-density difference on the lumenal

side of the membrane. Based on the X-ray structure,

this site is sandwiched between two lumenal

loops connecting M3/M4 and M7/M8. We propose

a mechanism for TG inhibition that involves

increased rigidity amongst the transmembrane

helices, thus preventing movements necessary for

binding Ca2..

Mechanism of TG inhibition

Given the exceedingly high af®nity of TG, our

proposed site is likely to prevent movement of

these two lumenal loops relative to one another.

Such movement may be required to achieve the

cooperative binding of two calcium ions and the

transition to the E1 state. Indeed, to optimize the ®t

of the atomic coordinates from the E1

_Ca2 state to

the EM map in the E2 state, the M7/M8 loop was

moved _10 AÊ closer to the M3/M4 loop. Therefore,

TG may be acting as a tether to prevent the

conformational transition to the E1 state. Furthermore,

since one full transport cycle can occur before TG inhibition, the two loops may not come

in close proximity until after Ca2. is released to the

lumen. Thus, movement of these loops in a bellows-

like fashion may be one of the key structural

changes during transport.

In addition to preventing Ca2. binding, TG

prevents the backward reaction with inorganic phosphate9 and drastically reduces the af®nity of

the E2 conformation for ATP,26 suggesting that TG

effects more than just the lumenal and transmembrane

domains. Such ATP binding by the E2 conformation

is known to accelerate the transition to

the E1 conformation and to affect the ion-binding

properties of Ca2.-ATPase27 and of Na.,K.-

ATPase. These ATP effects are certain to involve

some sort of conformational change, which may

well in¯uence the disposition of the lumenal loops.

By resisting these conformational changes, TG

could effectively lower the af®nity for ATP and

other ligands in distant domains. In particular, the

increased occupancy of decavanadate within the

cytoplasmic domain may re¯ect these long-range

effects of TG on the ATP-binding and phosphorylation

domains.

The effect of mutations in the S3 segment on TG

af®nity implicates this region in relaying the structural

changes between the distant binding sites. In

the E1

_Ca2 structure, this S3 segment is hydrogen

bonded to both the M6/M7 loop and S4, and is

certainly affected by the 90 _ rotation of the cytoplasmic

nose (yellow domain in Figure 3(c) and

(d)) during the E2 to E1 transition. The density for

M3 and S3 are quite well de®ned in the cryoEM

map of the E2 conformation and our ®tting of the

atomic coordinates required the continuous M3/S3

helix to be bent near the cytoplasmic membrane

surface. If S3 were acting as a lever arm to connect

cytoplasmic and lumenal domains, then mutations

that alter the local structure of S3 near this bend

could in¯uence the lumenal domains, thereby

indirectly affecting TG af®nity. Thus, TG inhibition

represents a speci®c example of the structural

coupling that generally characterizes Ca2.-ATPase

and, in fact, all P-type ATPases. Analogous

changes are likely to coordinate ion-binding in the

transmembrane domain with speci®c chemical

changes at the phosphorylation site, thus driving

the cycle of ATP-dependent ion transport.

NITRIC OXIDE

Beside its use as an experimental tool, thapsigargin possesses

several pharmacologically interesting properties. Since it is able to kill

slowly proliferating and non-proliferating cells, prodrugs of thapsigargin,

which are currently undergoing preclinical evaluation, have

been developed as novel means for anti-cancer therapy, notably for

the treatment of prostate cancer (Denmeade et al., 2003). Furthermore,

compounds disrupting the Ca2+ homeostasis in endoplasmic

reticulum (ER), including thapsigargin, have been shown to possess

virustatic activity. Thapsigargin inhibits production of infectious

cytomegalovirus (CMV) virions (Isler et al., 2005), replication of

hepatitis C virus (HCV) (Nakagawa et al., 2005), herpes simplex virus

(HSV) (Cheshenko et al., 2003), and Sendai virus (HVJ) (Ono and

Kawakita, 1994). It has been suggested that antiviral effects of

thapsigargin may depend on the activation of unfolded protein

response which is detrimental to viral infection (Isler et al., 2005;

Nakagawa et al., 2005). Anyhow several data can be regarded as a

sound argument for the possibility that activation of the immune

defence might be attributable to other, so far not revealed effector

mechanisms.

One of the major factors inhibiting replication of a number of

viruses via several plausible pathways encompassing inhibition of

ribonucleotide reductase activity, generation of tricarboxylic acid

cycle intermediates, and suppression of an intermediate-early

transactivator protein, Zta, is nitric oxide (NO) (Karupiah and Harris,

1995; Mannick et al.,1995; Melkova and Esteban,1995). NO is effective

against all poxviridae, herpetoviridae, rhabdoviridae, retroviridae, and

parvoviridae, including CMV (He et al., 1995), HCV (Sharara et al.,

1997), HSV (Nathan and Hibbs, 1991) and many others (Karupiah and Harris, 1997), although not all viruses including e.g. HVJ (Yoshitake

et al., 2004) are sensitive to the virucidal activity of NO in vitro.

Thapsigargin has been shown to interfere with nitric oxide (NO)

biosynthesis. The effects of thapsigargin on expression of inducible NO

synthase (iNOS)mRNA andNOproduction have frequently been studied

in combination with various NO primary activators such as lipopolysaccharide

(LPS) (Chen et al., 2005; Jordan et al., 1995; Kiemer and

Vollmar, 2001; Korhonen et al., 2001; Park et al.,1996; Park et al., 1995),

interferon-γ (IFN-γ) (Raddassi et al., 1994), and interleukin-β (IL-1β)

(Geng and Lotz,1995). The findings are controversial, showing both mild

stimulation (Jun et al., 1996; Kmoníčková et al., 2005; Park et al., 1996;

Park et al., 1995; Raddassi et al., 1994) and inhibition (Geng and Lotz,

1995; Chen et al., 2005; Jordan et al.,1995; Kiemer and Vollmar, 2001) of

NO in mouse peritoneal macrophages or mouse macrophage cell lines.

The reasons for the discrepancy are not clear but they seemto depend on

the type and intensity of cell stimulation (Kmoníčková et al., 2005;

Korhonen et al., 2001).

The aim of the present experiments was to analyze underlying

mechanisms determining the interference of thapsigargin with NO

biosynthesis. The attention has been focused on possible role of

cytokines, key activators of high-output NO production (Bogdan et al.,

1994). The findings demonstrate unequivocally that thapsigargin

activates secretion of IFN-γ in rat and mouse macrophages as well as

in human peripheral blood mononuclear cells.

In summary, the original data show unequivocally that thapsigargin

may be regarded as a novel immunostimulatory agent. It is a

potent activator of IFN-γ secretion in both animal and human cell

systems. Since endogenous IFN-γ has been implicated in control of

growth of an impressive range of obligate and facultative intracellular

organisms (Kaufmann, 1993), the results presented here suggest a

perspective for further preclinical evaluation of this agent.

SOCE

Store-operated Ca2+ entry (SOCE) is a ubiquitous signaling

mechanism in non-excitable and excitable cells that

is triggered by a reduction in the level of Ca2+ in intracellular

stores [1]. Physiologically, SOCE most commonly

results from stimulation of cell surface receptors that evoke

Ca2+ release through IP3 receptors or ryanodine receptors

in the ER, which then activates store-operated Ca2+ channels

(SOCs) in the plasma membrane (PM). By supplying a source

of Ca2+ for refilling stores, SOCE enables the ER to release

Ca2+ over extended periods and thereby evoke sustained Ca2+

oscillations, exocytosis, and other Ca2+-dependent events in

response to prolonged stimulation [1]. In addition, SOCE can contribute directly to the elevation of cytosolic Ca2+

levels ([Ca2+]i), as in T lymphocytes where SOCs generate

the sustained Ca2+ signals needed to drive gene expression

underlying T cell activation by antigen [2].

In the years following the original proposal of the

"capacitative Ca2+ entry" hypothesis by Putney [3], [Ca2+]i

measurements in a variety of cells treated with thapsigargin

(TG) and other SERCA inhibitors to deplete stores revealed

that SOCE was widespread [4]. However, because [Ca2+]i

is influenced by many factors in addition to channels (e.g.,

pumps, transporters, membrane potential, and organelles),

an essential step in defining the entry pathway for SOCE

was the identification of a store-operated Ca2+ current (the

Ca2+ release-activated Ca2+ current, or ICRAC) in mast cells

and T cells using patch-clamp techniques. The discovery of

the CRAC current progressed in stages, beginning with the

description of an extremely small Ca2+ current stimulated

by IP3 in mast cells [5] and by mitogens in Jurkat T cells

[6], and proceeding to the demonstration that it was store-dependent, based on activation by intracellular Ca2+ buffers

and ionomycin [7] and by thapsigargin [8].

The CRAC channel emerged as a prototypic SOC for several

reasons. First, it has fulfilled the most stringent criteria

for being store-operated, in that it is activated by a variety of

procedures that reduce free Ca2+ in the ER under conditions

of constant cytosolic [Ca2+] [1,9]. Second, its properties have

been extensively characterized, establishing a distinctive "fingerprint"

of the channel that later proved to be essential in

identifying the CRAC channel gene [9]. Finally, the CRAC

channel was found to have a critical physiological function,

in that a spontaneous loss of CRAC channel activity in T

cells causes a severe combined immunodeficiency (SCID)

syndrome in human patients [10-12].

Despite many efforts over the past 20 years to unravel the

mechanism underlying SOCE, relatively little progress was

made largely due to a complete lack of identified SOCE proteins

[9].However, the recent identification of genes encoding

the CRAC channel and the Ca2+ sensor in the ER has made

possible for the first time direct experiments that reveal the

activation process and the structure of the fundamental units

of SOCE. In this review we present a historical overview of

attempts to understand coupling between the ER and SOCs,

followed by a discussion of recent work describing the elementary

units of SOCE and their assembly in response to

store depletion.