Substantial overlap occurred between the TRPV1- and thapsigargin-sensitive Ca2+ pools, and TRPV1 immunofluorescence colocalized with the endoplasmic reticulum targeting motif "…KDEL…". To determine if TRPV1-induced release of Ca2+ from internal stores activated endogenous store-operated Ca2+ entry, the effect of 2-APB on Ba2+ was evaluated. 2-ABP blocked thapsigargin-induced Ba2+ influx, but not RTX-induced Ba2+ influx. In the combined presence of thapisgargin and RTX, the 2-APB sensitive component was essentially identical to the thapsigargin-induced component. These results indicate that TRPV1 forms agonist-sensitive channels in the endoplasmic reticulum, which when activated, release Ca2+ from internal stores but fail to activate

store-operated Ca2+ entry. 11

Maitotoxin (MTX), a potent marine toxin, is a tool for the study of a Ca2+- overload induced necrotic/oncotic cell death. Upon stimulation with MTX, bovine aortic endothelial cells (BAEC) undergo sequential changes in plasmalemmal permeability.

2+ Initially, MTX activates CaNSC leading to an increase in [Ca ]i. Second is the activation

of large pores known as cytolytic/oncotic pores (COP) that allows ions and small

molecules (< 800Da) to enter the cell. Finally, the cells lyse, not by plasma membrane

rupture but through the activation of a "death" channel. Concurrent with COP activation

is the formation of plasma membrane blebs, which dilate as the cell undergoes lysis.

Previous studies have shown that removal of extracellular Ca2+ or blockade of the MTX-

2+ induced rise in [Ca ]i block all subsequent steps in the cell death cascade suggesting one

2+ or more of the steps is dependent upon or modulated by the rise in [Ca ]i. The purpose of the study is to evaluate the role that Ca2+ plays in the regulation of the each phase of the

MTX-induced cell death cascade. The results indicate that Ca2+ influences the initiation of COP, the time to cell lysis, and membrane blebbing.

Chapter 1

Introduction

I . Ca2+ channels and endogenous Ca2+ signalling

A. Ion channels

Ion channels play an important role in physiological functioning in both excitable and nonexcitable cells. In excitable cells like neurons and cardiac cells, they are responsible for the maintenance of the resting membrane potential, the initiation and propagation of action potentials, and in cell to cell communications in the form of synaptic transmission. In nonexcitable cells ion channels play a role in maintaining the resting membrane potential, the regulation of cell volume, and cell communication and signaling. Ion channels primarily are characterized by two properties, permeation and gating (for review see (71)). Permeation refers to the movement of ions through the aqueous pore of the channel. Permeation can further be distinguished by the characteristics of conduction and selectivity (2). Selectivity refers to the type of ions that the channel conducts, such as cations (Na+, K+, Ca2+) or anions (Cl-). Some ion channels are highly selective for particular ions, whereas others display a lesser degree of selectivity allowing several types of ions to permeate their conducting pathway. The second ion channel characteristics, gating, refers to factors that influence channel opening and closure (2). Ion channels may be gated by a variety of stimuli, including electrical energy (voltage), thermal energy (heat and cold), chemicals (ligands, pH, toxins, phosphorylation, lipids), and mechanical stress (osmolarity and cell swelling, shear and pressure).

B. Endogenous Ca2+ signalling

The Ca2+ signaling cascade is a ubiquitous cellular mechanism by which external

signals control several crucial aspects of cell and tissue function (for review see

(4,24,140,150)). Most cells maintain a 10,000-fold concentration gradient between the

extracellular and intracellular Ca2+ environment (10-3 versus 10-7 M, respectively). This

concentration gradient is maintained by the actions of ATP-driven Ca2+ pumps capable of

pumping Ca2+ against its concentration gradient. These pumps are localized in both the

plasma membrane and endoplasmic reticulum. The endoplasmic reticulum is a

quantitatively important source of Ca2+, with an estimated free [Ca2+] of a few hundred micromolar (43). Stimulation of G-protein coupled receptors at the plasma membrane linked to phospholipases (PLC-β/γ) leads to hydrolysis of phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2) and the generation of inositol (1,4,5)-triphosphate (IP3) and diacylglycerol (DAG) (140). The lipid diacylglycerol activates protein kinase C (PKC) causing phosphorylation of target proteins (127). Inositol (1,4,5)-triphosphate diffuses to the cytosol and activates IP3 receptors (IP3R) on the endoplasmic reticular membrane causing a release of Ca2+ from endoplasmic reticulum Ca2+ stores (6). Ca2+ released into the cytosol following IP3R activation is returned to the ER via by the

sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) (107). Ca2+ is also pumped

out of the cell by the plasma membrane Ca2+ ATPase (PMCA) (187). Additionally, for

the cell to maintain proper Ca2+ homeostasis, it becomes necessary for the cell to refill depleted Ca2+ stores from a source of Ca2+ outside of the cell. This is accomplished by channels in the surface membrane activated following depletion of intracellular Ca2+ stores. Thapsigargin, a specific high affinity inhibitor of SERCA pumps, depletes the 15

internal Ca2+ store and concomitantly activates Ca2+ entry in the absence of measurable

phosphoinositide hydrolysis (188). Thus, store depletion per se appears to be sufficient

to trigger activation of Ca2+ influx via surface membrane channels. These channels,

thought to be gated by the filling state of intracellular Ca2+ stores, are known as store- operated channels. The mechanism by which depleted Ca2+ stores are refilled is known as store-operated Ca2+ entry (SOCE).

While the existence of SOCE is well established and ubiquitously observed, the

exact mechanism of activation remains controversial. There are three hypotheses

regarding how the depleted Ca2+ stores communicate with surface membrane channels in

SOCE; conformational coupling, a diffusible messenger, and membrane fusion. (For review see (43,140,151)) In the conformational coupling hypothesis (85), there is physical association between IP3R in the ER and store-operated channels on the plasma membrane. Depletion of intracellular Ca2+ stores leads to an alteration in this coupling that activates surface membrane store-operated channels. Activation of these store- operated channels on the plasma membrane leads to Ca2+ influx and the refilling of depleted Ca2+ stores. In the diffusible messenger hypothesis, the store depletion results in the release of a yet unidentified cellular factor that diffuses from the ER to the plasma membrane to activate surface membrane SOC channels (152). The final proposed mechanism for SOCE is the membrane fusion hypothesis. This hypothesis suggests that store-operated channels sequestered into membrane vesicles that are released and inserted into the plasma membrane following depletion of intracellular stores (49,219). There is evidence in the literature that supports each of these hypotheses, however the exact mechanism responsible for the activation of surface membrane channels following 16

depletion of intracellular Ca2+ stores remains controversial. Although the exact molecular identity of store-operated channels is unknown, some members of the transient receptor

potential (TRP) family of Ca2+ permeable, nonselective cation channels have been

implicated as possible candidates (for review see (26,68,118).

C. The TRP superfamily of ion channels

The TRP family of ion channels are Ca2+-permeable nonselective cation channels.

In general, these channels poorly discriminate between Na+ and K+ and have varying permeability to Ca2+. Members of the TRP superfamily of ion channels function in a

variety of roles related to sensory transduction (25). The founding member of the TRP

family of ion channels was named for a gene involved in Drosophila phototransduction

(66). These ion channels are responsible for the PLC-dependent Ca2+ influx that occurs following light stimulation. A mutation of this channel, known as transient receptor potential, causes a transient, as opposed to a sustained Ca2+ influx in response to light stimulation (28,119,120). The trp gene encodes a protein with structural similarity to known ion channels including voltage gated potassium channels, cyclic nucleotide-gated channels (145), and hyperpolarization-gated cation channels. TRP channel family members have 6 hydrophobic membrane-spanning regions, a pore-loop structure between

TM5 and TM6, and intracellular N- and C-termini. In addition TRP channels also contain

3-4 ankryin repeats located at the NH2-terminus. These proteins are also distinguished by the a region of high sequence homology located just downstream of the 6th transmembrane region. This 25 amino acid region contains a proline rich domain and an invariant region known as the TRP box (Glu-Trp-Lys-Phe-Ala-Arg) (118). Similar 17

voltage-gated potassium channels (101,108), cyclic nucleotide-gated channels (204), and

hyperpolarization activated channels (217), TRP channels are thought to be functionally

assemble as tetramers (78,93,97). Mammalian TRP homologues comprise 3 main

families. These include the classical or canonical TRPC's, the melastatin-like TRPM, and

the vanilloid-like TRPV's. Although the relative sequence similarity between families is

low, these proteins are grouped based upon their structural similarity and common

function as Ca2+-permeable nonselective cation channels (CaNSC).

There are seven reported mammalian genes that encode for members of the canonical or TRPC family. These proteins like their Drosophila counterparts share 6 hydrophobic transmembrane regions and pore-loop structure, 3-4 ankryin repeats, and

TRP domain. TRPC family members form receptor operated channels linked through activation of G-protein coupled receptors and receptor tyrosine kinases coupled to PLC

(15,77,223). They are abundantly expressed in brain, but are also found in most peripheral tissues (52,154). Members of the TRPC family co-associate along specific lines. TRPC1, 4, and 5 form heteromeric complexes both in vivo in brain and when heterologously expressed (55,78,180). TRPC3, 6, and 7 form heteromeric channels and are all are activated by DAG (25,77). TRPC2 is a pseudogene in humans, although it is thought to be expressed in the vomeronasal organ of rats (100). Due to their regulation by PLC and their Ca2+ permeability, TRPC channels were candidate store operated channels activated in response to depletion of intracellular Ca2+ stores (26,68,118).

The melastatin-like or TRPM family is comprised of eight members. These

proteins share common structure with classical TRP including the presence of the TRP

box but do not possess ankryin repeats (119). The founding member is known as TRPM1 18

or melastatin. TRPM1 is downregulated in high grade metastatic melanoma tumors in

mice (41). TRPM2, expressed in brain microglia and cells of the immune system (95), is

activated by ADP-ribose, NAD, and H2O2, and thus may play a role as a cellular redox

sensor (25,144). This channel is unique due to the presence of a carboxy-terminal ADP-

ribose pyrophosphatase linked to the channel (144). TRPM3 is activated by hypo-

osmolarity and is expressed in the kidney and CNS (58). Unlike other TRPM family

2+ members, TRPM4 is highly permeable to monovalent ions and is activated by [Ca ]i (KD

~400 nM) (98). TRPM4 is thought to cause membrane potential depolarization following receptor-mediated Ca2+-release from internal stores (98). This then modulates the driving force for Ca2+ entry to refill depleted stores. TRPM5 is expressed primarily in taste receptors responsible for bitter and sweet stimuli (143). Like TRPM4, TRPM5 conducts primarily monovalent cations and is activated by Ca2+ (76). Two TRPM family members,

TRPM6 and TRPM7, are unique due to functional kinase domains located in their carboxy-termini (156). TRPM6 is a Mg2+ permeable channel responsible for intestinal

and renal Mg2+ reabsorption (207). TRPM7 is a ubiquitously expressed CaNSC that is

2+ inhibited by ~0.6 mM [Mg ]i, and inactivates following PIP2 hydrolysis (157). In addition, TRPM7 has been shown to play a key role in ROS-induced Ca2+ entry in anoxic neuronal cell death (1). In fact, blockade or suppression of TRPM7 expression prevents anoxic neuronal cell death (1). The menthol receptor, TRPM8, is activated by menthol and cold temperatures (> 25°C), and is expressed predominately in sensory neurons, the trigeminal ganglion, and prostate (114).

The final subfamily of the TRP superfamily of ion channels is the vanilloid or

TRPV family. This family contains 6 members. The founding member TRPV1, or 19

vanilloid receptor, is activated by compounds containing vanilloid moieties, acidic pH,

and high temperatures (> 42° C) (19). TRPV1 is involved in the sensing and transduction of painful stimuli, and is expressed primarily in sensory neurons (19). TRPV2, also known as VRL-1 or GRC, is a capsaicin-insensitive TRPV1 homologue with a higher temperature threshold of activation (> 53°C) (18). TRPV3, also a TRPV1 homologue, is expressed in keratinocytes and has a lower threshold of activation (> 30°C) (172,216).

There is a report that TRPV1 and TRPV3 coimmunoprecipitate raising the possibility that these channels form heteromultimers in vivo with intermediate thermal thresholds (216).

The osmosensory protein TRPV4 senses changes in cell volume and hypo-osmolarity, and is activated by phorbol esters and endocannibinoids (179,208,211). The final two members of the TRPV family are the epithelial Ca2+ entry proteins commonly know as

CaT and eCaC. TRPV5, also known as CaT2 and eCaC1, is involved in Ca2+ uptake by the kidney (73,206). TRPV6, also known as CaT1 and eCaC2, is involved in intestinal

Ca2+ absorption (142). These epithelial Ca2+ channels are highly similar electrophysiologically, with only subtle differences in sensitivity to the blocker ruthenium red and in Ca2+-dependent regulation (74). TRPV5 and TRPV6 have also been reported to co-assemble as functional heteromultimers (75). In addition, there is a

2+ report that TRPV6/CaT1 manifests the pore properties of ICRAC, the Ca -release- activated-current observed following depletion of intracellular Ca2+ stores (222).

D. The vanilloid receptor type 1, TRPV1

It is well established that treatment of sensory neurons with capsaicin, the active

ingredient in chili peppers, leads to Ca2+ influx (19,38,109,215). Recently, the gene for 20

the protein responsible for this activity, TRPV1, was cloned from dorsal root ganglia

(19). The vanilloid receptor (TRPV1) is a ligand-gated Ca2+-permeable nonselective cation channel. Expression of TRPV1 is found primarily in peripheral pain sensing neurons and spinal cord lamina involved in the transmission of painful stimuli (116). A

TRPV1 knock-out mouse (VR1-/-) showed severely deficient pain sensation when stimulated with vanilloids, pH and heat (17). Capsaicin, the active ingredient in chilli peppers, is a high affinity agonist of the vanilloid receptor. Resiniferatoxin, derived from a cactus-like plant Euphorbia resinifera, acts as an ultrapotent vanilloid agonist (182).

Other stimuli known to activate TRPV1 include both natural and synthetic compounds with vanilloid moieties, acidic pH (< 5.5) (191), lipids including the cannibinoid agonist anandamide (133) and eicosanoid derivatives (83), and high temperature (>42°C)

(19,191). In addition, N-arachidonoyl-dopamine has been shown to act as a potent endogenous capsaicin-like activator of TRPV1 (82). Antagonists of TRPV1 include capsazepine (8), indo-resiniferatoxin (163), SB-366791 (63), BCTC (201), and

ruthenium red (19).

The regulation of TRPV1 is complex. In addition to the multiple modes of stimuli

known to lead to its activation, TRPV1 sensitization occurs through several mechanisms.

Sensitization of TRPV1 plays an important role in the increased sensation of painful

stimuli during inflammation (164). Many inflammatory mediators can lower the

activation threshold of TRPV1 for its stimuli. Stimulation of VR1 expressing cells with

agents known to activate phospholipase-C beta (β) and gamma (γ)-linked pathways can

potentiate TRPV1 responses to agonist, heat, and pH. These agents include ATP (P2Y1)

(192), bradykinin (Gq/11/PLCβ)(23,147,181), and NGF (TRK-A/PLC-γ)(13,23). The 21

mechanism, whether direct PKC phosphorylation of the channel, or release of latent

inhibition by phosphatidylinositol 4,5-bisphosphate hydrolysis remains controversial.

One group has reported that PIP2 binds to the C-terminus of TRPV1 inhibiting its gating

(148). Disruption of this interaction through mutagenesis, application of an anti-PIP2 antibody, or cleavage of PIP2 by PLC activation, all lead to potentiation of responses to

chemical and thermal stimuli (23). Several other groups have reported that the

potentiation observed following activation of PLC is via direct phosporylation of TRPV1

by PKC. (130,134,147,181,205). Bradykinin is capable of TRPV1 sensitization through

activation of the phosholipase A2- lipoxygenase pathway via 12-HPETE (83,84). Mildly acidic stimuli (pH ~ 6.2) potentiates the response to capsaicin and lowers the threshold of activation for heat and agonists (89,191). TRPV1 undergoes Ca2+-dependent

desensitization that can be overcome by simultaneous application of multiple stimuli

(191). Channel phosporylation with cAMP-dependent protein kinase (PKA) has been

reported to reduce TRPV1 desensitization (9), and potentiate the response to capsaicin

(209) and anandamide (32). In addition, TRPV1 has been shown to bind calmodulin, and

disruption of this interaction prevents desensitization (129,155). Phosphorylation of

TRPV1 with CaMKII (92) and PI3K (13) are involved in TRPV1 activation by both

agonists and heat; while dephosphorylation of TRPV1 with the phosphatase calcineurin

induces desensitization (36). Based upon its mechanism of activation and regulation,

TRPV1 is an important integrator of painful stimuli.

Recent studies suggest that TRPV1 is tetrameric (93,97), but the actual subunit

composition or the ability of TRPV1 to form heteromultimers with other members of the

TRP channel family remains largely unknown. However, a recently cloned member of 22

the TRPV family, TRPV3, associates with TRPV1 when heterologously expressed, and

both TRPV1 and TRPV3 are co-expressed in dorsal root ganglion cells consistent with

heteromultimeric subunit assembly (172). Although there is little evidence in the

literature that TRPV1 channels can be activated by depletion of internal Ca2+ store per se,

Olah et al. (135) report that activation of heterologously expressed TRPV1 in COS-7

2+ cells and native TRPV1 in dorsal root ganglion cells, gives rise to an elevation of [Ca ]i in the absence of extracellular Ca2+. Since the binding site for agonist agents on the

channel is thought to be cytoplasmic (31,88,90,91) and TRPV1 agonists are highly

lipophilic, this result suggests that both heterologously-expressed and native TRPV1

channels may exist in the ER and when activated, initiate the release of Ca2+ from internal stores. Alternatively, agonist stimulation of plasmalemma-associated TRPV1 may activate PLC and initiate the release of stored Ca2+. The ability of TRPV1-induced

Ca2+ release to activate SOCE however, remains unknown (Fig. 1.1). The experiments described in chapter 2 were designed to evaluate the interaction of the vanilloid receptor

(TRPV1) with endogenous Ca2+ signaling mechanisms.

II. The role of Ca2+ channels in necrotic/oncotic cell death

A. Role of TRPV1 in cell death

Previous studies have shown that application of capsaicin to dorsal root ganglion

neurons causes neurotoxicity (22,72). This capsaicin-induced neurotoxicity is caused by

Ca2+ influx through TRPV1 as blockade with ruthenium red or removal of extracellular

Ca2+ blocks capsaicin-induced neurotoxicity in vivo (22). In addition agonist activation of 23

heterologously expressed TRPV1 causes cytotoxicity is several cell types (135).

Treatment with TRPV1 antagonists capsazepine or ruthenium red blocks agonist-induced

cytotoxicity in HEK cells heterologously expressing TRPV1 (57). These results suggest

2+ that increases in [Ca ]i following agonist stimulation of TRPV1 are capable of causing a

Ca2+-overload induced cytotoxicity. In addition, TRPV1-mediated release of Ca2+ from

2+ internal stores could provide an additional pathway for elevation of [Ca ]i in response to

stimulation. The release of Ca2+ from intracellular stores could provide a mechanism for

nociceptor desensitization and subsequent cell death that occurs following prolonged

stimuli (19,183).

B. Ca2+ overload and cell death

2+ A rise in [Ca ]i has been shown to cause cytotoxcity in a variety of cells and

tissues (136) and is a common signal in both apoptotic and necrotic cell death (137,220).

2+ 2+ Elevations in [Ca ]i can occur as the result of Ca influx across the plasma membrane or

2+ 2+ the release of Ca from intracellular stores. This increase in [Ca ]i has been shown to

disrupt intracellular signaling and mitochondrial function, alter protein phosphorylation,

generate reactive oxygen species, and cause activation of endonucleases, phospholipases,

and proteases (96,220). The ultimate death pathway, whether by an apoptosis or necrosis,

depends upon the strength and duration of the insult (220). Apoptosis, or programmed

cell death, is characterized by caspase activation, cell shrinkage, chromatin condensation,

and retention of membrane integrity (34,111,139). Apoptosis occurs following DNA

damage, growth factor deprivation, oxidative insult, or as the result of a genetic program

(34,221). Pro-apoptotic signals can be received through cell surface receptors (extrinsic 24

pathway) or through the release of molecules from the mitochondria following cell stress

(intrinsic pathway) (70,70).

Necrosis, or pathological cell death results from more severe and acute insults

which cause increases in membrane permeability, cell swelling, and membrane blebbing

(111,139,196). Necrotic cell death occurs after oxidative insult resulting from ischemia-

reperfusion injury in the brain following stroke and myocardium following infarction

(139,160). Oxidant stress constitutes reactive oxygen species (ROS) generated as a result

of arachidonic acid metabolism, the synthesis of nitric oxide, and uncoupling of electron

transport in the mitochondria. Following injury, there is excessive generation of free

-• radicals containing unpaired electrons like superoxide anion (O2 ), hydroxyl radical

• • (OH ), and nitric oxide (NO ) (16). Other molecules, like hydrogen peroxide (H2O2), peroxynitrite (ONOO-) do not have free radicals per se but act as oxidizing agents (16).

2+ Oxidant stress has also been shown to produces excessive increases in [Ca ]i (20) and can activate CaNSC (173).

Necrosis is thought to occur as a consequence of ATP depletion (139). In ischemia, disruptions of blood flow lead to a decrease in the delivery of oxygen to tissues, reduced availability of ATP and phosphocreatanine, and a switch to anaerobic metabolism (169). The decline in ATP reduces ability of ATP-driven ion pumps and exchangers on both the endoplasmic reticulum and plasma membrane resulting in a disruption of normal ion gradients. This cellular loss of ionic homeostasis has two consequences. Failure of the Na+-K+ ATPase leads to Na+ influx, osmotic imbalance, and water flow into the cell. This water influx causes cellular swelling common in necrosis and can ultimately cause swelling-induced cell lysis known as oncosis. In addition, the 25

accompanying membrane depolarization and loss of both the ER and plasma membrane

2+ 2+ 2+ Ca -ATPase function further increase [Ca ]i (169). This increase in [Ca ]i has been shown to disrupt intracellular signaling and mitochondrial function, alter protein phosphorylation, generate reactive oxygen species, and cause activation of endonucleases, phospholipases, and proteases (96,220). While the hallmarks of necrotic cell death have been well described (disruption of ion homeostasis, cellular swelling, increased membrane permeability, and membrane blebbing), little is known about how the rise in Ca2+ is coupled to the events in necrotic cell death. Thus, it is important to

establish the mechanism(s) involved in linking the Ca2+ overload to increases in membrane permeability, membrane blebbing, and cell lysis observed in necrosis.

C. Maitotoxin

Natural products and toxins (e.g., cholera toxin, pertussis toxin, tetrodotoxin,

digitalis, ryanodine, thapsigargin) represent powerful tools for the identification and

characterization of specific biochemical pathways important for cell signaling.

Maitotoxin, a potent marine toxin isolated from the dinoflagellate Gambierdiscus toxicus,

is the causative agent of ciguatera seafood poisoning. Stimulation with maitotoxin (MTX)

2+ increases [Ca ]i in all cells examined to date (64). In addition, MTX causes

neurotransmitter and hormone release, phosphoinosotide breakdown, contraction of

smooth, skeletal, and cardiac muscle, and calpain activation (64,210). There is mounting

evidence that MTX acts as a high affinity agonist for a yet unidentified receptor (112).

When added to liposomes or mitochondria, MTX does not possess ionophore activity

(122,185). In addition, trypsinization of human skin fibroblasts eliminates MTX-induced 26

Ca2+ influx suggesting that proteins at the plasma membrane may be involved in the response to MTX (65). Thus, these facts, coupled with the low nanomolar potency and saturability suggest that MTX may act as a high affinity agonist for a yet unidentified receptor. Several groups suggest that MTX binding to its proposed receptor is dependent upon extracellular Ca2+ (64,112,121), although there is a report of MTX-activated Na+ channels in guinea-pig ventricular cells in the absence of extracellular Ca2+ (126). MTX- induced increases in Ca2+ are also blocked by a variety of inorganic (Co2+, Mn2+, La3+)

(64) and organic (imadozole and SKF96365) channel blockers (30,174). While the identity of the MTX receptor remains unknown, several reports demonstrate that MTX activates Ca2+-permeable nonselective cation channels in a variety of cell types

(30,35,44,112,122,123,160). Because of its ability to activate CaNSC leading to a

2+ 2+ massive increase in [Ca ]i, MTX is a valuable tool for the study of Ca -overload induced cell death.

D. The Maitotoxin induced cell death cascade in endothelial cells

Because of its location and function, the endothelium is a major target of oxidant

stress (105). The endothelium plays a crucial role in normal cardiovascular functioning.

The endothelium is involved in regulation of blood coagulation and the immune

response, angiogenesis, blood-tissue permeability and transport, and the regulation of

vascular tone. Insults like those that occur in ischemia-reperfusion compromise the

metabolic state of the endothelium, lead to the generation of reactive oxygen species, the

disruption of endothelial cell function (105), and ultimately can cause a Ca2+ overload induced cell death (96). 27

The MTX-induced cell death cascade in bovine aortic endothelial cells (BAEC)

is a useful in vitro model of Ca2+-overload induced necrotic/oncotic cell death (45).

Treatment of BAEC cells with MTX leads to three sequential changes in plasma membrane permeability (Fig. 1.2). Initially, stimulation with MTX causes the activation

2+ 2+ of CaNSC, causing a massive increase in [Ca ]i. The rise in [Ca ]i occurs in a graded fashion with a EC50 for MTX of ~0.3 nM (45). As seen in Fig. 1.2 (blue box) the rise in

2+ 2+ [Ca ]i can be monitored using the fluorescent Ca indicator fura-2. Following the activation of CaNSC, there is an opening of large endogenous pores in the plasma membrane. Once activated, these pores allow ions and small molecules (< ~800 Da) to permeate the membrane (160,161). The opening of plasma membrane pores disrupts normal ionic gradients by allowing the influx of Na+, Ca2+, and Cl-, and the efflux of K+.

These ionic rearrangements cause water flow into the cell, changes in osmotic pressure, cell swelling, and ultimately cellular lysis (45). Thus, these pores have been termed cytolytic/oncotic pores (COP) (161).

Changes in plasma membrane permeability can be monitored through the use of ethidium- and propidium-based vital dyes. Ordinarily these dyes are excluded from the cytosol, however, upon pore formation or activation, the dyes are able to permeate the plasma membrane, bind nucleic acids and fluoresce Fig. 1.2 (orange box). Stimulation of

BAEC cells with MTX results in a biphasic uptake of ethidium. The initial (slow phase) of EB uptake represents COP activation/formation and the second (faster phase) represents cell lysis. Previous studies using dyes of increasing molecular weight have shown that the rate of dye uptake in the initial phase is inversely proportional to dye molecular weight, but the second phase is all-or-nothing and independent of molecular 28

weight (45,47). Thus, the initial phase (COP) likely represents the activation of a pore of

fixed size and defined permeability (45); whereas the second final, all-or-nothing phase

of the cell death cascade is the activation of a "death" channel or lytic pore (47).

Activation of the lytic pore can be monitored in cell populations by the release of large

cytoplasmic proteins like lactate dehydrogenase (MW 140 kDa), and at the single cell

level by the release of cell-associated green fluorescent protein (MW 28 kDa) (47).

Additionally, the uptake of propidium iodide (MW 668), which has a low permeability

through COP, can be used to monitor cell lysis at both the population and single cell level

(47) (Fig. 1.2, purple box). Blockade of the lytic pore can be achieved through the use of

the cytoprotectant amino acids glycine and L-alanine (47). These amino acids block the

cell lysis in a concentration-dependent manner (EC50 = ~1mM) without affecting the

2+ MTX-induced rise in [Ca ]i or COP activation. Blockade with these agents is reversible and stereospecific, as L-alanine is an effective blocker whereas D-alanine is ineffective.

In addition to the observed changes in plasma membrane permeability, there is a

dramatic change in cell morphology in the form of membrane blebs. These blebs are

small spherical protrusions from the plasma membrane ~3-5 µm in diameter that form concurrently with COP and proceed to dilate during cell lysis. Blebbing occurs when actin attachments to the underlying cytoskeleton are weakened (5). Evidence in the literature has shown that the interactions between actin and myosin is an important mediator of membrane blebbing (193). Additionally, both myosin light chain phosphorylation (117) and Rho kinase I activation (27) are required for apoptotic blebbing. Membrane blebbing has been previously described in cells undergoing both apoptosis (27,117) and necrosis (45,202). Apoptotic and necrotic blebbing can be 29

distinguished by both their kinase dependency and growth characteristics. While both

types of blebs exhibit similar neck diameters (~ 1µm) at the point of attachment to the plasma membrane, they differ by pattern of growth. Necrotic blebs grow indefinitely whereas apoptotic blebs shrink or retract following formation (5), a process often referred to as cytokinesis or zeosis (46). Apoptotic bleb are also far more sensitive to kinase inhibitors compared to necrotic membrane blebs. Inhibition of p38MAPK, Rho kinase 1, and MLCK all inhibit the formation of membrane blebs while having little effect upon necrotic blebbing (5,27).

The MTX-induced cell death cascade occurs in a variety of cell types including human embryonic kidney cells, THP-1 and BAC1 macrophages, human skin fibroblasts,

Chinese hamster ovary, and rat insulinoma cell lines (160,161). In addition, the MTX- induced cell death cascade is virtually identical to both oxidant stress- and P2X7-induced cell death (160,161). This suggests that the events in this cascade may represent a common, conserved, biochemical pathway that can be initiated through the activation of different Ca2+ permeable nonselective cation channels. Previous studies have shown that

2+ 2+ removal of extracellular Ca or blockade of the MTX induced rise in [Ca ]i blocks the subsequent steps in the cell death cascade (30,174). In addition, blockade of the MTX- induced Ca2+ influx with U73343 blocks the progression of cell death and rescues BAEC cells from oncosis (46). This suggests that steps in the cell death cascade are dependent

2+ upon or modulated by a rise in [Ca ]i. While the steps in the MTX-induced cell death cascade are well established, the specific role(s) that Ca2+ plays in the initiation and progression of steps in the cell death cascade remain largely unknown. The experiments 30

described in Chapter 3 are designed to evaluate the role of high affinity Ca2+ binding proteins in each phase of the MTX-induced cell death cascade.

Fig. 1.1. Intracellular expression of TRPV1 and potential influence on store-operated

Ca2+ entry. Schematic representation of how TRPV1 expressed on the endoplasmic

reticulum may interact with endogenous store operated Ca2+ entry mechanisms. Proposed store-operated Ca2+ entry mechanisms (see text for discussion): conformational coupling

(I), diffusible messenger (II), and membrane fusion (III). TRPV1 channels (red) are expressed on both the plasma membrane and endoplasmic reticulum. Abbreviations: store-operated channel (SOC), endoplasmic reticulum (ER), and Inositol 1,4,5 phosphate receptor (IP3R).

III.

C II. O C

S O S

P (+)

ER I. ? Ca2+ C O S ? Ca2+

Store-operated Ca2+ entry mechanisms I: Conformational coupling II: Diffusable messenger III: Membrane fusion

Fig. 1.2. The maitotoxin-induced cell death cascade in bovine aortic endothelial cells.

Schematic representation of changes in plasmalemmal permeability and cellular

morphology following MTX stimulation of bovine aortic endothelial cells. Highlighted

boxes show the sequential changes in membrane permeability following MTX

stimulation of endothelial cells suspended in buffer. MTX-induced Ca2+ entry is monitored with fura-2 (blue box); COP activity as monitored via uptake of ethidium bromide (orange box), and cell lysis is monitored via the uptake of propidium iodide

(purple box). See figure 3.1 legend for further description and chapter 3 for discussion.

Ethidium Bromide Uptake

Fura-2 Propidium Iodide Fluoresence Uptake

COP CaNSC Lytic pore MTX Receptor?

Ca2+ EB ? PI/GFP ? ?

Bleb formation Bleb dilation 35

Chapter 2

Activation of vanilloid receptor type I (TRPV1 channel) in the

endoplasmic reticulum fails to activate store-operated Ca2+

entry

INTRODUCTION

The purpose of the present study was to evaluate the interaction of TRPV1 with

endogenous cellular signalling mechanisms. Specifically, the experiments were designed

to determine if TRPV1, a) is activated by depletion of internal Ca2+ stores per se, b) causes the release of Ca2+ from internal stores, and c) activates or inhibits endogenous

SOCE. To accomplish these goals, the activity of TRPV1 was measured following

heterologous expression in Sf9 insect cells using recombinant baculovirus. This

eukaryotic expression system has several unique features that that are advantageous for

the expression of channels and proteins involved in Ca2+ signal transduction. First,

following infection with recombinant baculovirus, host cell protein synthesis is decreased

within 6 hours and is essentially zero after 24 hours (132). Since heterologous protein

expression is under control of the polyhedrin promoter, a late turn-on promoter in the

baculovirus life cycle, problems associated with co-expression or upregulation of host-

cell proteins are minimized and the likelihood of recording homomeric channel activity is

increased. Second, Sf9 cells exhibit G-protein-coupled Ca2+ signaling essentially identical to that observed in mammalian cells, including thapsigargin-induced SOCE

(79). Lastly, Sf9 cells lack endogenous Ca2+-activated channels that may contribute to the Ca2+ signaling profile observed in other expression systems such as HEK cells or

Xenopus oocytes (7981,200). The results of the present study demonstrate that TRPV1, functionally expressed in Sf9 insect cells, shares pharmacological characteristics of both the native channels found in nerve and TRPV1 channels heterologously expressed in mammalian cells. However, the results also show that activation of TRPV1 causes an 37

2+ 2+ increase in [Ca ]i that reflects both the release of Ca from internal stores and the influx

of Ca2+ from the extracellular space. Furthermore, although the TRPV1- and

thapsigargin-sensitive internal Ca2+ pools substantially overlap and TRPV1 immunoreactivity co-localizes with a marker for the ER, stimulation of TRPV1 alone failed to activate endogenous SOCE. When TRPV1 and SOCE were simultaneously activated, cation influx was essentially additive suggesting that TRPV1 in the plasmalemma and SOCE are functionally independent. The results are consistent with the hypothesis that only a specific fraction of the ER is linked to SOCE. Furthermore, the selective activation of TRPV1 present in the ER by its endogenous ligand, may play an important role in Ca2+ signal transduction within specific subcellular microdomains. This

chapter has been previously published as: Wisnoskey BJ, Sinkins WG, and Schilling WP

Biochem J 372:517-526,2003.

MATERIAL AND METHODS

Solutions and reagents. MES buffered saline (MBS) for use with Sf9 cells contained

the following: 10mM CaCl2, 60mM KCl, 17mM MgCl2, 10mM NaCl, 4mM D-glucose,

110mM sucrose, 0.1% bovine serum albumin (BSA), and 10mM MES, pH adjusted to

6.2 at 22oC with NaOH. The total osmolarity of the MES-buffered saline was ~340

2+ mOsm. Nominally Ca free MBS was identical to MBS with the exception that CaCl2 was isosmotically replaced by MgCl2. Hepes-buffered saline (HBS) for use with mammalian cells contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM D-glucose,

o 1.8 mM CaCl2, 15 mM HEPES, and 0.1% BSA, pH adjusted to 7.40 at 37 C with NaOH.

2+ Ca -free HBS contained the same salts as HBS without added CaCl2. Rabbit and guinea pig anti-VR1 antibodies were obtained from Chemicon International (Temecula, CA).

Mouse anti-KDEL was from Stressgen Biotechnologies (Victoria, BC Canada).

Rhodamine Red-X-conjugated donkey anti-Mouse IgG and FITC-conjugated donkey anti-Guinea Pig were from Jackson ImmunoResearch Laboratories (West Grove, PA).

Fura-2/AM was obtained from Molecular Probes (Eugene, OR). Capsaicin and capsazepine were obtained from Calbiochem (San Diego, CA), and resiniferatoxin (RTX) from Alexis Corporation (San Diego, CA).

Cell culture. Spodoptera frugiperda (Sf9) cells were obtained from the American Type

Culture Collection (Rockville, MD) and cultured as previously described (80,132) in

Graces insect medium supplemented with 2mM L-glutamine, 1% penicillin- 39

streptomycin-neomycin solution (PSN), 10% heat-inactivated fetal bovine serum (FBS),

2% yeastolate solution, and 2% lactalbumin hydrosylate solution (Gibco, Grand Island,

NY). Human embryonic kidney cells (HEK), obtained from ATCC, were maintained

with minimum essential medium (MEM) supplemented with 10% FBS, 1% PSN, and 2

mM L-glutamine. For passage, HEK cells were dispersed by trypsin treatment and

seeded to a density of ~3 × 103 cells/cm2. The medium was changed every 2-3 days following seeding.

Generation of a HEK cell line stably expressing TRPV1. The cDNA encoding rat

VR1 (TRPV1), a generous gift from Dr. David Julius (University of California, San

Francisco), was subcloned into a bicistronic EGFP-containing vector pIRES-EGFP,

yielding pIRES-EGFP-TRPV1. Wild-type HEK cells were transfected using FugeneTM transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturers protocol. Briefly, 35 mM dishes of wild-type HEK cells at ~95 % confluence were incubated with serum-free MEM. Fugene (2 µl) and pIRES-EGFP-

TRPV1 cDNA (~1 µg) were allowed to complex in 100 µl of serum-free MEM in a polystyrene tube. After 20 minutes, this complex was added to the HEK cells and allowed to incubate for 24-48 hrs. Cells were dispersed with trypsin and sparsely seeded onto 100mm dishes with MEM containing ~200 µM G418. Following selection,

individual colonies were assessed visually using fluorescence microscopy. Green

fluorescent colonies were isolated using plastic cloning rings, dispersed with trypsin and

re-seeded onto new dishes with fresh media containing G418. Individual colonies were

grown to confluence and assayed for functional TRPV1 expression by fura-2 40

fluorescence assay. To increase the level of TRPV1 expression in the stably-expressing

o HEK cells, confluent dishes were maintained at 26 C in a 5% CO2 atmosphere for 96 hrs prior to performing experiments.

Generation of recombinant baculovirus. The cDNA encoding TRPV1 was subcloned into a baculovirus transfer vector, pVL1393 using standard techniques. Recombinant baculovirus was produced using the BaculoGoldTM Transfection Kit (Pharmingen, San

Diego, CA) as described in the instructions provided by the manufacturer. Recombinant viruses were amplified to obtain a high titer viral stock solution. The virus was stored at

4oC under sterile conditions and used for infection of Sf9 cells as described previously

(79,80).

Infection of Sf9 insect cells with recombinant baculovirus. Sf9 cells in Grace’s medium were plated onto 100mm culture dishes (~105 cells/cm2). Following incubation for 30 min, an aliquot of viral stock was added (multiplicity of infection ~10) and the cells were maintained at 27oC in a humidified air atmosphere. Unless otherwise indicated, cells were used at 24-28 hrs post-infection.

Isolation of membrane-associated TRPV1 protein. Sf9 cells were harvested at various

postinfection times, subjected to centrifugation at 500 × g for 5 min, and resuspended at a density of 5 × 106 cells/ml in lysis buffer containing 20 mM Tris-Cl, 5 mM EDTA, 1 mM

EGTA, and 1 mM phenylmethylsulfonyl fluoride. The cell suspension was sonicated on

ice three times for 10 s with a 10 s rest between pulses using a Sonic Dismembranator 41

(Fisher) on a power setting of 2.5. The cell lysate was subjected to centrifugation at 6000

× g for 10 min at 4 oC. The resulting pellet was discarded and the supernatants were centrifuged at 42,000 × g for 30 min. The microsomal pellets were resuspended in lysis buffer at protein concentration of 5-10 mg/ml and stored at –80 oC until use. Protein concentration was determined by the method of Lowry using BSA as the standard (104).

Immunoblotting of TRPV1 Protein. An equivalent volume of 2x sample buffer (60

mM Tris-Cl, pH 6.8, 2% SDS, 10 % glycerol, 100 mM dithiothreitol, and 0.025%

bromphenol blue) was added to an aliquot of membrane preparation and the reaction was

incubated at 100 oC for 1 min. Protein samples (20 µg) were subjected to electrophoresis on 7% polyacrylamide gels along with molecular weight standards (Bio-Rad). Proteins were transferred electrophoretically from the polyacrylamide gels to Millipore polyvinylidene difluoride membranes (100V for 1hr on ice). Blots were probed with an anti-capsaicin receptor (TRPV1) polyclonal antibody at a dilution of 1:3000 for 1 h at room temperature. After washing, membranes were probed for 1 h with goat anti-rabbit

IgG and detected using ECL-Plus (Amersham Life Sciences).

Immunolocalization of TRPV1 Protein. Sf9 cells were plated onto polylysine-coated glass coverslips and subsequently infected with recombinant baculovirus containing VR1.

Following incubation for 28 hrs at 27oC, the coverslips were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 mins. Following fixation, coverslips were washed 3 times with PBS, and permeabilized by incubation for 5 minutes in PBS 42

containing 0.1% Triton X-100. Coverslips were blocked with 4% non-fat dry milk/PBS

for 1 hr and incubated overnight at 4oC with primary antibodies diluted 1:1000 (guinea pig anti-VR1) or 1:200 (mouse anti-KDEL) in 4% milk/PBS solution. Coverslips were subsequently washed 3 times with PBS and incubated with FITC-conjugated donkey anti- guinea pig IgG (1:200) and/or Rhodamine REDX-conjugated donkey anti-mouse IgG

(1:500) for 1 hour at room temperature. Following incubation with secondary antibodies, the coverslips were washed 3 times and mounted onto glass slides using Vectashield

(Vector Laboratories Inc., Burlingame, CA) mounting medium.

Confocal microscopy. Immunofluorescence and DIC images were obtained as single scans using a Leica TCS SP2 confocal microscope and a 100x oil-immersion objective.

Dual imaging of FITC and Rhodamine REDX were obtained by sequential scans to

minimize cross excitation. Two scans were averaged for each image.

2+ 2+ 2+ Measurement of cytosolic free Ca concentration ([Ca ]i). [Ca ]i was measured in

Sf9 cells using the fluorescent indicator, fura-2, as previously described (79,80). Briefly,

Sf9 cells were dispersed, washed and resuspended at a concentration of 1.5-2 x 106 cell/ml in MBS containing 2 µM fura-2/AM. Following 30 min incubation at room temperature (22oC), the cell suspension was subjected to centrifugation, resuspended in an equal volume of MBS and incubated for an additional 30 min. For experiments performed on HEK cells, dispersed cells were suspended in HBS containing 20 µM fura-

2/AM. After a 30-min incubation at 37 oC, the cell suspension was diluted 10-fold with

HBS, incubated for an additional 30 minutes, washed, and resuspended in fresh HBS. 43

The cells were twice washed immediately before measurement and fluorescence was

monitored using an SLM 8100 spectrophotofluorometer. Excitation wavelength

alternated between 340 and 380 nm and fluorescence intensity was monitored at an

emission wavelength of 510 nm. All measurements on Sf9 cells were performed at 22oC and HEK experiments were performed at 37 oC. Calibration of fura-2 associated with the

cells was accomplished using Triton X-100 lysis in the presence of a saturating

2+ 2+ concentration of Ca followed by addition of EGTA (pH 8.5). [Ca ]i was calculated by

2+ the equation of Grynkiewicz et al. (60) using a Kd value for Ca binding to fura-2 of 278 nM for 22oC (168) and 224 nM for 37 oC. For experiments using the Ca2+ surrogate Ba2+, fluorescence intensity was measured at an emission wavelength of 510 nm and excitation wavelengths of 350 and 390 nm as previously described (159). The results shown are mean traces from "n" number of independent experiments. At selected time points, the mean ± standard error values are plotted as symbols in each figure.

RESULTS

Functional expression of TRPV1 in Sf9 insect cells. TRPV1 was heterologously

expressed in Sf9 insect cells using recombinant baculovirus. Protein expression was

driven by the polyhedrin promoter, a late turn-on promoter in the baculovirus life cycle.

Membrane-associated TRPV1 protein was monitored by Western blot analysis using an

anti-capsaicin receptor polyclonal antibody. The predicted molecular weight of TRPV1

is 95 kDa. TRPV1 protein was non-detectable at 0 and 12 hrs, but increased in a time-

dependent fashion from 18 to 48 h post-infection time (Fig 2.1A), a profile consistent

with expression of a protein under control of the polyhedrin promoter. To determine if

the TRPV1 protein formed functional channels, TRPV1-expressing Sf9 cells (TRPV1-

cells) were loaded with the fluorescent Ca2+ indicator, fura-2, and subsequently

2+ challenged with capsaicin. Capsaicin (1 µM) had no effect on [Ca ]i at 0 and 12 hrs post-infection time. However, stimulation with 1 µM capsaicin produced a time-

2+ dependent increase in [Ca ]i from a resting level of ~150 nM to peak values of 0.5 µM,

1.5 µM, and 2.5 µM at 18, 28 and 48 hrs post-infection time, respectively (n=3; Fig

2+ 2.1B). Capsaicin had no effect on [Ca ]i in uninfected Sf9 cells or in Sf9 cells infected with an unrelated baculovirus, i.e., cells infected with recombinant baculovirus containing the M5 muscarinic receptor cDNA (see below). These results demonstrate that

TRPV1 activity directly correlates with protein expression. All subsequent experiments

were performed at 24-28 h post-infection time.

Characterization of TRPV1 expressed in Sf9 insect cells. TRPV1 expressed in

mammalian cells can be stimulated by 1) capsaicin, 2) resiniferatoxin, a high affinity

toxin isolated from Euphorbia plants (19), and 3) anandamide, the putative endogenous

cannabinoid receptor agonist (224). RTX, capsaicin, and anandamide each produced a

2+ concentration-dependent increase in [Ca ]i in TRPV1-expressing Sf9 cells with EC50 values of 166 pM, 24.5 nM, and 3.89 µM, respectively (Fig 2.2). For each

2+ concentration of capsaicin and anandamide tested, [Ca ]i increased to a peak value within 1 min and remained at a sustained level for the duration of the recording. In

2+ contrast, the rate of change of [Ca ]i following the addition of RTX, increased as

2+ function of RTX concentration, i.e., both the peak level and the time-to-peak [Ca ]i changed as a function of RTX concentration. Thus, although RTX is the more potent agonist, the kinetics of TRPV1 activation by RTX differ from those of capsaicin and anandamide. To determine if TRPV1 expressed in Sf9 cells could be blocked by capsazepine, a known competitive antagonist of TRPV1 in mammalian cells, the response to either capsaicin or anandamide was measured in the absence and presence of increasing concentrations of capsazepine. Capsazepine produced a concentration- dependent inhibition of the TRPV1-induced response (data not shown).

Effect of TRPV1 stimulation on internal Ca2+ stores. To determine if stimulation of

TRPV1 causes release of Ca2+ from internal stores, capsaicin and RTX were added to

TRPV1-cells suspended in Ca2+-free extracellular buffer. Concentrations of capsaicin

2+ (<100 nM) and resiniferatoxin (<1 nM) that produced dramatic increases in [Ca ]i in the presence of extracellular Ca2+, had little or no effect in the absence of Ca2+. However, 46

higher concentrations of both capsaicin and RTX produced a graded release of Ca2+ from internal stores (Fig 2.3A and 2.3B). The kinetic differences between capsaicin and RTX observed in the presence of extracellular Ca2+ (see Fig 2.2), were also evident in the

absence of extracellular Ca2+ (Fig 2.3), i.e., capsaicin produced an immediate increase in

2+ [Ca ]i, whereas the response to RTX was more slowly developing suggesting a different mechanism of receptor activation for the these two agonists.

Upon binding to specific plasmalemmal receptors, many agonist agents cause the release of Ca2+ from internal stores through a G-protein-coupled activation of PLC and

2+ subsequent generation of Ins(1,4,5)P3. The ability of capsaicin and RTX to release Ca from internal stores may reflect either the presence of TRPV1 in the ER or the activation of PLC. To determine if TRPV1-mediated release of Ca2+ from internal stores is secondary to the activation of PLC, the effect of U73122, an inhibitor of PLC and

2+ U73343, the inactive analog (189), on TRPV1-dependent change in [Ca ]i in the absence

of extracellular Ca2+ was examined. As seen in Fig 2.3C, U73122 blocked the carbachol-

2+ induced release of Ca from internal stores in control Sf9 cells expressing the M5

2+ muscarinic receptor, but had no effect on the RTX-induced release of [Ca ]i from internal stores in TRPV1-cells (Fig 2.3D). U73343 had no effect on the agonist-induced response in either cell type. Thus, the ability of TRPV1 agonists to release Ca2+ from

internal stores is unrelated to PLC activity and probably reflects the presence of

functional TRPV1 channels in the ER. To determine if TRPV1 is present in the ER,

confocal images of TRPV1-expressing Sf9 cells, co-labeled with antibodies directed

against TRPV1 and antibodies against the ER targeting motif KDEL (128), were 47

examined. The nucleus occupies a large proportion of the cytoplasm in Sf9 cells and the

ER is seen in a narrow region surrounding the nucleus (Fig 2.4B). Although the level of

TRPV1 immunofluorescence was variable from cell to cell, there was substantial co-

localization of TRPV1 to the ER (Fig 2.4C).

Effect of TRPV1-induced Ca2+ release on store-operated Ca2+ entry. In essentially all cells examined to date, depletion of the internal Ca2+ store by thapsigargin, an inhibitor of SERCA pumps, is linked to activation of Ca2+-permeable cation channels in the plasma membrane (140). The resultant store-operated Ca2+ entry (SOCE) gives rise

2+ 2+ to a long-lasting elevation of [Ca ]i. In most cell types, SOCs are permeable to Sr and

Ba2+ and inhibited by low micromolar concentrations of La3+ and Gd3+. Lanthanides are

also potent inhibitors of endogenous SOCE in Sf9 insect cells (79,81). To determine if

depletion of internal Ca2+ stores per se, activates TRPV1, thapsigargin-induced Ba2+ influx in control M5-expressing Sf9 cells was compared to that observed in TRPV1-cells.

As seen in Fig 2.5A, addition of thapsigargin to control Sf9 cells in the absence of

extracellular Ca2+ caused a release of Ca2+ from internal stores. Subsequent re-addition of Ba2+ produced an increase in fluorescence ratio that was blocked by the presence of 10

µM La3+, indicative of Ba2+ influx via the SOCE mechanism. Addition of RTX (100

2+ 2+ nM) had no effect on basal [Ca ]i or on basal Ba influx in control M5-expressing cells

(Fig 2.5B). Thus, as expected RTX has no significant effect on thapsigargin-induced

Ca2+ signaling in the absence of TRPV1 expression. We next examined Ba2+ influx in

TRPV1 cells in response to thapsigargin and RTX. As seen in Fig 2.5C, expression of

TRPV1 had no significant effect on the ability of thapsigargin to release Ca2+ from 48

internal stores or on the subsequent La3+-inhibitable Ba2+ influx component. In contrast,

addition of 1 nM RTX to TRPV1-cells produced a dramatic increase in Ba2+ influx, that

was relatively insensitive to the presence of La3+ (Fig 2.5D). This concentration of RTX

did not release Ca2+ from internal stores. These results demonstrate that TRPV1

expression has no effect on endogenous SOCE, and that TRPV1 is not itself a store-

operated channel.

Effect of 2-APB on Ba2+ influx via SOCE and TRPV1. To determine if TRPV1- mediated Ca2+ store release activates endogenous SOCE, we examined the effect of 2- aminoethoxydiphenyl borate (2-APB), a known inhibitor of SOCE in mammalian cells

(14,86,141,162,162). These experiments were performed in a paired fashion on the same batch of TRPV1-infected Sf9 cells to eliminate problems associated with variation in

TRPV1 channel expression levels. As seen in Fig 2.6A, 2-APB (10 µM) greatly attenuated thapsigargin-induced Ba2+ influx in TRPV1-expressing Sf9 cells. The effect was concentration dependent and no further inhibition was observed at concentrations above 10 µM (data not shown). We next compared the effect of 2-APB on Ba2+ influx induced by either a non-store-releasing (1 nM) or a store-releasing (100 nM) concentration of RTX (Fig 2.6B and 2.6C). In the absence of 2-APB, Ba2+ influx was identical in response to either concentration of RTX. Thus, the release of stored Ca2+ observed at the higher concentration of RTX does not appear to activate an additional component of Ba2+ influx related to SOCE. Consistent with this hypothesis, 2-APB had no effect on Ba2+ influx at either concentration of RTX. To detemine if RTX releases

Ca2+ from a thapsigargin-sensitive compartment, the two agonists were sequentially 49

added to TRPV1 expressing cells in the absence of extracellular Ca2+. As seen in Fig 2.7,

the RTX-releasable pool was greatly reduced by prior treatment with thapsigargin.

Likewise the thapsigargin-releasable component was reduced by prior stimulation with

RTX. Overall, the results confirm that activation of TRPV1 channels in the ER releases

Ca2+ from a thapsigargin-sensitive Ca2+ pool, but fails to activate endogenous SOCE.

Effect of TRPV1 activation in the ER on SOCE. It is possible that activation of

TRPV1 in the thapsigargin-sensitive pool prevents or inhibits activation of endogenous

SOCE. To test this hypothesis, the effect 2-APB on Ba2+ influx was examined following the release of Ca2+ from internal stores by the sequential addition of RTX and

thapsigargin. As seen in Fig 2.8A, addition of a non-store-releasing concentration of

RTX (1 nM) did not prevent thapsigargin-induced Ca2+ release and a substantial

component of Ba2+ influx seen under this condition was inhibited by the presence of 2-

APB (Fig 2.8B). The 2-APB-sensitive component was essentially the same in cells

stimulated with a store-releasing concentration of RTX (100 nM; Fig 2.8C and 2.8D).

These results demonstrate that activation of TRPV1 does not prevent or inhibit activation

of endogenous SOCE.

Stimulation of HEK cells stably expressing TRPV1. The baculovirus/Sf9 cell

expression system is commonly used to obtain high levels of protein expression. Since

TRPV1 agonists are lipophilic, and the ligand binding site on TRPV1 is thought to be

cytoplasmic (88,90,91), we considered the possibility that functional TRPV1 retained in

internal membrane as a result of high expression in Sf9 cells, may give rise to agonist- 50

induced release of Ca2+ from internal stores. A recent study found evidence for the

presence of functional TRPV1 channels in the ER of COS-7 cells transiently expressing

TRPV1 and in cultured primary DRG neurons (135). We therefore, re-examined the

effect of capsaicin and RTX on fura-2-loaded HEK cells stably expressing TRPV1. As

seen in Fig 2.9A and 2.9B, RTX and capsaicin caused a time- and concentration-

2+ dependent increase in [Ca ]i with an EC50 value of 52 pM and 221 nM, respectively. In

comparison to Sf9 cells, these values are 3-fold lower for RTX, but 10-fold higher for

capsaicin (see above). Kinetically however, the responses to capsaicin and RTX were

virtually identical in HEK cells compared to Sf9 cells. Capsaicin produced an immediate

response at each concentration tested, whereas the time-to-peak was inversely related to

2+ RTX concentration. Notably, both capsaicin and RTX produced an increase in [Ca ]i in the absence of extracellular Ca2+ (Fig 2.9C and 2.9D), consistent with the hypothesis that

these agents release Ca2+ from internal stores. No effect of either RTX or capsaicin on

2+ [Ca ]i was observed in non-transfected HEK cells (data not shown). Thus, the response observed is dependent upon TRPV1 expression. Despite the difference in apparent affinities of TRPV1 for RTX and capsaicin, the overall profile is remarkably similar between HEK and Sf9 cell expression systems. Furthermore, these results are consistent with TRPV1 localization to both internal and surface membranes.

We next examined the effect of 2-APB on Ca2+ influx induced by depletion of the stores by either thapsigargin or RTX. 2-APB attenuated thapsigargin-induced Ca2+ entry

(Fig 2.10A), but had no significant effect on RTX-induced Ca2+ influx (Fig 2.10B),

2+ although there was a tendency for [Ca ]i to decline to a lower level at longer times after 51

re-admission of Ca2+ to the cuvette in the presence of 2-APB. These results suggest that

endogenous SOCE in HEK cells is not activated by RTX-induced release of Ca2+ from

internal stores. To determine if TRPV1 activation inhibits endogenous SOCE, the effect

of 2-APB was examined following the sequential release of Ca2+ stores by RTX and thapsigargin (Fig 2.10C). As was seen in the Sf9 cell, a substantial component of Ca2+ influx was blocked by 2-APB under this condition. The magnitude of the 2-APB- sensitive component seen in Fig 2.10C was only slightly reduced from that seen in Fig

2.10A. Thus, Ca2+ influx via surface membrane TRPV1 and endogenous SOCE are

essentially additive suggesting that the two pathways are completely independent.

Importantly, the results show that RTX-induced Ca2+ release in HEK cells stably

expressing TRPV1 fails to activate endogenous SOCE (Fig 2.10B). 52

DISCUSSION

In the present study, TRPV1 was functionally expressed in Sf9 insect cells and

HEK cells. TRPV1 expressed in both cell types exhibited pharmacological

characteristics similar to those reported for native channels and for TRPV1

heterologously expressed in other cell types. Specifically, TRPV1 was activated by RTX,

capsaicin, and anandamide with the appropriate potency order and the response was

antagonized by capsazepine. However, slight differences in agonist potencies were

45 2+ noted. Based on Ca uptake in neurons, mast cells and glioma cells, the EC50 for capsaicin and RTX ranged from 300 to 700 nM and from 0.5 to 2.1 nM, respectively

(183). A survey of the literature shows that the EC50 values for capsaicin and RTX in heterologous expression systems ranged from 33 to 1000 nM and from 0.1 to 39 nM, respectively (19,57,82,87,135,165,175,191,212). In the present study, TRPV1 expressed in HEK cells shows a similar agonist sensitivity (221 and 0.05 nM for capsaicin and

RTX, respectively). However in Sf9 cells, the apparent affinity for capsaicin was increased 10-fold to 24 nM whereas the affinity for RTX was reduced 3-fold to 0.16 nM.

There are 3 major difference between the Sf9 and HEK cell experiments. First, the activity of TRPV1 in the Sf9 cells most likely reflects homomeric channels, whereas in

HEK cells it is possible that other members of the TRP channel family, endogenously expressed in this cell line, may contribute subunits to the functional channels observed.

Second, the Sf9 cell experiments were performed at 22oC, whereas the HEK experiments

were at 37oC. Although TRPV1 channel activity appears to be relatively insensitive to changes in temperature over this range (19), the EC50 for capsaicin was reported to 53

change from 125 to 18 nM in stably expressing HEK cells when temperature was reduced

from 37 to 22oC (175). Third, the HEK experiments were performed at pH of 7.4,

whereas the Sf9 cell buffers have a pH of 6.2. It is well established that reducing the pH

increases sensitivity of TRPV1 to agonist agents (191). Thus, the differences in

temperature and pH could explanation the increase in the apparent capsaicin affinity in

Sf9 cells relative to HEK cells seen in the present study. The reason for the decrease in

RTX potency in Sf9 versus HEK is unknown. However, the kinetics of channel

activation by capsaicin and RTX are the same in both cell types. Capsaicin produced a

2+ 2+ rapid increase in [Ca ]i and the time to peak [Ca ]i was independent of concentration.

2+ In contrast, RTX produced a more slowly rising increase in [Ca ]i and the time to peak

was inversely related to RTX concentration. Similar profiles have been previously noted

(170,184), but the underlying mechanisms for the kinetic difference remains unknown.

Overall however, the general pharmacological profile for TRPV1, the relative potencies

for agonists, and the kinetics of channel activation for TRPV1 expressed in Sf9 cells is

similar to native channels and to channels heterologously expressed in other cell types.

Since large amounts of protein can be generated using this expression system, the Sf9 cell

may be useful for the ultimate biochemical purification of the channel in sufficient

quantities for structural studies.

When TRPV1-expressing cells were challenged with either RTX or capsaicin in

the absence of extracellular Ca2+, we observed a concentration-dependent increase in

2+ [Ca ]i. This increase was unrelated to activation of PLC since U73122, an inhibitor of

PLC, had no effect on RTX or capsaicin-induced mobilization of intracellular Ca2+. The 54

immunohistochemical results of the present study confirm localization of TRPV1

channels to the ER. Furthermore, the RTX-sensitive internal stores partially, but

substantially overlapped the thapsigargin-sensitive pool. Together the results suggest that

TRPV1, present in the ER is functional and can be activated by exogenously applied

lipophilic agonists. However, in stark contrast to thapsigargin, RTX-induced release of

Ca2+ from internal stores failed to activate 2-APB-sensitive cation influx. This result was obtained in both Sf9 cells and in HEK cells stably expressing TRPV1. Furthermore, experiments in which stores were depleted by sequential addition of both thapsigargin and RTX demonstrated that SOCE was not inhibited by activation of TRPV1 in the ER.

Since the TRPV1 and thapsigargin-sensitive pools substantially overlap, yet only thapsigargin activates SOCE, it seems reasonable to conclude that there must be an internal store that is not "linked" to SOCE. A similar conclusion was recently derived

2+ from studies of the Ca release activated current, ICRAC, in TRPV1-transfected RBL

cells.

TRPV1-induced release of Ca2+ from internal stores has been reported in dorsal root ganglion cells in vivo (102,135) and TRPV1-transfected cells in vitro (110,135,199).

These investigators suggested that the mobilization of Ca2+ via ER-localized TRPV1

could explain effects of capsaicin in neurons that appeared to be independent of

extracellular Ca2+ (149). In preliminary studies, anandamide (in the absence of extracellular BSA) caused a concentration-dependent (10-30 µM) release of Ca2+ from internal stores (data not shown). Thus compartmentalized production of an endovanilloid such as anandamide or n-arachidonoyl-dopamine (NADA) (82) may give rise to Ca2+ signaling events localized to specific subcellular microdomains. There is growing 55

evidence that members of the TRP channel family are held together in large multimeric

signaling complexes that may contain other proteins and enzymes critical to the signal

transduction cascade (e.g., PLC, PKC, calmodulin). Furthermore, the "signalplex" may

be localized to specific subcellular domains by PDZ-containing scaffolding proteins such the Drosophila protein responsible for the inactivation-no-afterpotential D mutant

(INAD) or the Na+/H+-exchanger regulatory factor (NHERF) (118). In this regard,

Delmas et al (33) showed that TRPC1 over-expressed in superior cervical ganglion neurons appears to be localized to a specific microdomain that includes a plasmalemmal bradykinin receptor and an endoplasmic reticulum Ins(1,4,5)P3 receptor. This localization of TRPC1 appears to be important for channel activation by depletion of the internal Ca2+ store. In platelets it appears that TRPC6 is localized to the plasmalemma, whereas TRPC1 is found in intracellular membrane compartment (69). There is also growing evidence for functional heterogeneity of Ca2+ stores in neurons (146), but the

role of TRP channels in these stores remains unknown. Compartmentalized production

of anandamide or NADA along with selective localization of TRPV1 to a specific ER

microdomains in neurons may play an important role in "intracellular" Ca2+ signaling events without involvement of plasmalemmal Ca2+ channels.

FIGURES

Fig. 2.1. Time course of TRPV1 expression in Sf9 insect cells. Panel A. Membrane preparations were isolated as described in Materials and Methods at 0 (uninfected Sf9 cells), 12, 18, 28 and 48 hrs after infection with recombinant baculovirus containing

TRPV1 cDNA. Protein expression was determined by Western blot using rabbit anti-

TRPV1 polyclonal antibody. Panel B. Sf9 cells were infected with TRPV1 baculovirus.

At the indicated post-infection times, the cells were harvested, loaded with fura-2, and suspended in MES-buffered saline (MBS). Five traces are shown superimposed. At the time indicated by the arrow, capsaicin (1 µM; in this and all subsequent figure legends, final concentrations are indicated) was added to the cuvette and the fluorescence recorded. The lines shown are mean values from 3 independent experiments. Symbols represent mean ± SE values at selected time points.

Fig. 2.2. Concentration-response relationship for TRPV1 agonists. Fura-2-loaded

2+ Sf9 cells expressing TRPV1 were suspended in MBS. Agonist-induced change in [Ca ]i following receptor stimulation was determined for various concentrations of capsaicin

[Panel A; 10 µM (z), 1 µM (c), 100 nM (T), 10 nM (V), and 1 nM ()], RTX [Panel

B; 100 nM (z), 10 nM (c), 1 nM (T), 100 pM (V), 10 pM ()], and anandamide

[Panel C; 10 µM (z), 5 µM (c), 2 µM (T), 1 µM (V), and 100 nM ()].The lines shown are mean values from 3 independent experiments. Symbols represent mean ± SE values at selected time points. Panel D; Mean normalized peak-response at each

concentration was fitted to a binding equation. The best-fit apparent K0.5 values were determined for RTX (z; 166 pM), capsaicin (; 24.5 nM), and anandamide (S; 3.89

µM).

Fig. 2.3. Agonist-induced release of Ca2+ from internal stores in TRPV1-expressing

Sf9 cells. TRPV1-expressing Sf9 cells were suspended in nominally Ca2+ free MBS.

Cells were stimulated at the indicated time by capsaicin [Panel A; 100 µM (z), 10 µM

(c), 1 µM (T), and 100 nM (V)] or, RTX [Panel B; (10 µM (z), 100 nM (c),10 nM

(T), and 1 nM (V)]. Panel C. Sf9 cells expressing the M5 muscarinic receptor were

suspended in nominally Ca2+ free MBS. At the time indicated, carbachol (100 µM) was added in the presence of U73122 (10 µM; open circles) or U73343 (10 µM; closed

circles) added at 50 sec. Panel D. Sf9 cells expressing TRPV1 were suspended in

nominally Ca2+ free MBS. At the time indicated, RTX (100 nM) was added in the presence of U73122 (20 µM; open squares) or U73343 (20 µM; closed squares) added at

50 sec. The lines shown are mean values from 3 independent experiments. Symbols represent mean ± SE values at selected time points.

Fig. 2.4. Localization of TRPV1 to the ER. TRPV1-expressing Sf9 cells were co- labeled with TRPV1-specific antibodies and a monoclonal antibody directed against the

ER retention motif, KDEL as described in Material and Methods. The stained cells were visualized using confocal microscopy. Panel A. FITC-fluorescence (green) associated with TRPV1. Panel B. Rhodamine REDX fluorescence (red) associated with KDEL.

Panel C. Overlay of the fluorescence images shown in A and B. Panel D. Overlay of the fluorescence images with the DIC image. No fluorescence was observed in the absence of primary antibody, i.e., secondary only. Yellow color in C and D indicate areas of co-localization. Results are representative of two independent experiments.

A B

C D

Fig. 2.5. Effect of thapsigargin and RTX on Ba2+ influx in control and TRPV1- expressing Sf9 cells. Two mean traces are shown superimposed in each panel. Panels A and B. Fura-2-loaded control Sf9 cells (i.e., Sf9 cells infected with recombinant baculovirus containing the M5 muscarinic receptor cDNA) were suspended in nominally

Ca2+-free MBS in the absence (z) or presence () of 10 µM La3+. In each experiment,

Ba2+ influx through plasmalemmal channels was estimated from the fluorescence change following the addition of Ba2+ (10 mM) at the indicated time. Cells were treated with thapsigargin (Panel A, 200 nM, at 100 s) or RTX (Panel B, 100 nM, at 100 s). Panels C

and D. TRPV1-expressing Sf9 cells, loaded with fura-2, were treated with thapsigargin

(200 nM) or RTX (1 nM). The lines shown are mean values from 3 independent

experiments. Symbols represent mean ± SE values at selected time points.

Fig 2.6. Effect of 2-APB on thapsigargin- and RTX-induced Ba2+ influx in TRPV1- expressing Sf9 cells. TRPV1-expressing Sf9 cells were suspended in nominally Ca2+ free MBS. Panel A. Two mean traces are shown superimposed. In one, 2-APB (10 µM)

was added at the time indicated followed by thapsigargin (100 nM) and Ba2+ (10 mM).

Panel B. Four mean traces are shown superimposed. RTX, either 1 nM (squares) or 100 nM (circles) was added in the absence (open symbols) or presence (closed symbols) of 2-

2+ APB (10 µM). Panel C. The mean [Ba ]i was calculated from the data in Panel B following the addition of Ba2+ to the cuvette. Experiments are paired i.e., performed consecutively on the same batch of cells on the same day. The lines shown are mean values from 3 independent experiments. Symbols represent mean ± SE values at selected time points.

Fig. 2.7. Overlap of RTX- and thapsigargin-sensitive Ca2+ stores. TRPV1-expressing

Sf9 cells were suspended in nominally Ca2+ free MBS. Thapsigargin (200 nM) and RTX

(100 nM) were added to the cuvette at the times indicated by the arrows. The lines shown are mean values from 3 independent experiments. Symbols represent mean ± SE values at selected time points.

Fig. 2.8. Effect of 2-APB on thapsigargin-induced Ba2+ influx in absence or presence of RTX-induced store release in TRPV1-expressing Sf9 cells. Same experimental protocol as described in the legend to Fig 2.6, with the exception that thapsigargin was added after either 1 nM RTX (Panel A) or 100 nM RTX (Panel C) in the absence (open

2+ circles) or presence (closed circles) of 2-APB (10 µM). Panel B and D. Mean [Ba ]i was calculated from the data in Panels A and B, respectively, following the addition of

Ba2+ to the cuvette. Also shown is the 2-APB-sensitive component (∆) which was obtained by subtracting the Ba2+ influx in the presence of 2-APB from that obtained in

the absence of 2-APB. Experiments are paired i.e., performed consecutively on the same

batch of cells on the same day. The lines shown are mean values from 3 independent

experiments. Symbols represent mean ± SE values at selected time points.

Fig. 2.9. Concentration-response relationship for TRPV1 agonists in HEK cells

stably expressing TRPV1. Fura-2-loaded HEK cells stably expressing TRPV1

suspended in HBS were treated with capsaicin [Panel A; 10 µM (z), 1 µM (c), 100 nM

(), 10 nM ( ), and 1 nM (S)] and RTX [Panel B; 100 nM (z), 10 nM (c), 1 nM (),

100 pM ( ), 10 pM (S), 1 pM (U), and 100 fM (T)]. Panel C and D; HEK cells were

suspended in nominally Ca2+-free HBS and treated with capsaicin [Panel C; 100 µM (z),

10 µM (c), 1 µM (), and 100 nM ( )] or, RTX [Panel D; 1 µM (z), 100 nM (c), 10 nM (), 1 nM ( ), 100 pM (S), and 10 pM (U)].The lines shown are mean values from

3 independent experiments. Symbols represent mean ± SE values at selected time points.

Fig. 2.10. Effect of 2-APB on thapsigargin- and RTX-induced Ca2+ influx in

TRPV1-expressing HEK cells. TRPV1-expressing HEK cells were suspended in

nominally Ca2+ free MBS. Panel A. Two mean traces are shown superimposed. In one,

2-APB (50 µM) was added at the time indicated followed by thapsigargin (100 nM) and

Ca2+ (10 mM). Panel B. RTX (100 nM) was added in the absence (open circles) or presence (closed circles) of 2-APB (50 µM). Panel C. RTX (100 nM) and thapsigargin

(100 nM) were added at the times indicated in the absence (open circles) or presence

(closed circles) of 2-APB (50 µM). Experiments are paired i.e., performed consecutively on the same batch of cells on the same day. The lines shown are mean values from 3 independent experiments. Symbols represent mean ± SE values at selected time points.

Chapter 3

Maitotoxin-induced cell death cascade in bovine aortic endothelial cells: Divalent cation specificity and selectivity

INTRODUCTION

2+ Pharmacological blockade of the MTX-induced rise in [Ca ]i, in a variety of

cell types blocks many of the downstream cellular events associated with MTX toxicity

(30,174). In BAECs, pharmacological blockade of MTX-induced Ca2+ influx blocks the

uptake of vital dyes and rescues BAECs from oncosis (46). These results suggest that

2+ one or more steps in the cell death cascade require a rise in [Ca ]i. However, the exact

2+ step(s) that is sensitive to a rise in [Ca ]i is completely unknown. The fact that MTX-

induced cell death reflects a cascade of cellular events, each presumably dependent on the

previous step, and each potentially with specific requirements for Ca2+, poses a unique

challenge. For example, it is difficult to determine the effect of Ca2+ on a downstream

event such as membrane blebbing or cell lysis when an upstream step in the cascade, such

2+ as COP activation, may also require a rise in [Ca ]i. Therefore, in order to determine

2+ which step in the cascade is dependent on a rise in [Ca ]i we took advantage of the well-

known difference in affinities of various Ca2+-binding proteins for Ca2+, Sr2+, and Ba2+.

In particular, the affinity of Ca2+ binding proteins of the EF-hand type for Ba2+ is

approximately 2 to 3-orders of magnitude less than that for Sr2+ or Ca2+ (61). Therefore,

the involvement of a high affinity Ca2+ binding protein at a specific point in the cell death

cascade can be revealed by evaluation of divalent cation selectivity of the individual

steps. Experiments were performed at the population level to evaluate the average

response of the cells, and at the single cell level to correlate changes in plasmalemmal

permeability with changes in bleb formation and dilation. Additionally, the single cell

experiments provide important information concerning the heterogeneity, i.e., cell-to-cell

variability of the response. The results show for the first time that Ca2+ affects three

specific steps in the death cascade: 1) Ca2+ affects the kinetics of COP activation or

formation, 2) Ca2+ dramatically affects the time to cell lysis suggesting that a high affinity Ca2+ binding protein may be required for the opening of the death channel, and 3)

Ca2+ plays a fundamental role in bleb morphology. These results provide important clues

2+ to understanding how a rise in [Ca ]i controls events at the plasmalemma that ultimately lead to cell demise. The data presented in this chapter will been published as: Wisnoskey

BJ, Estacion MR, and Schilling WP AJP Cell Physiology 2004 (in press).

MATERIALS AND METHODS

Solutions and reagents. Normal HEPES-buffered saline (HBS) contained 140 mM

NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM D-glucose, 15 mM HEPES,

0.1% bovine serum albumin, pH adjusted to 7.4 at 37oC with NaOH. The composition of

2+ 2+ Ba -HBS and Sr -HBS was identical to that of HBS with the exception that CaCl2 was

iso-osmotically replaced with BaCl2 or SrCl2, respectively. Fura-2 acetoxymethyl ester

(fura-2/AM), BAPTA acetoxymethyl ester (BAPTA/AM), ethidium bromide (EB) and propidium iodide (PrI) were obtained from Molecular Probes (Eugene, OR). Ionomycin was from Calbiochem (San Diego, CA). Maitotoxin (MTX) was obtained from Wako

Bioproducts (Richmond, VA) and was stored as a stock solution in ethanol at –20oC. All

other salts were of reagent grade.

Cell Culture. BAECs were cultured as previously described (159) using Dulbecco's

modified Eagles medium (GIBCO) supplemented with 10% fetal bovine serum (Hyclone,

Logan UT), 100 µg/ml streptomycin, 100 µg/ml penicillin and 2 mM glutamine

(complete-DMEM). When grown to confluence, the cultures demonstrated contact- inhibited cobblestone appearance typical of endothelial cells.

2+ 2+ Measurement of the apparent cytosolic free Ca concentration. [Ca ]i was

measured using the fluorescent indicator, fura-2, as previously described (159).

Experiments were performed with cells in the twelfth to twentieth passage and 2-3 days

post-confluency. Briefly, cells were harvested and re-suspended in HBS containing 20 µ

M fura-2/AM. Following 30 min incubation at 37oC, the cell suspension was diluted

~10-fold with HBS, incubated for an additional 30 min, washed and resuspended in fresh

HBS. Aliquots from this final suspension were subjected to centrifugation and washed

twice immediately prior to fluorescence measurement. Fluorescence was recorded in a

mechanically stirred cuvette using an SLM 8100 spectrophotofluorometer. For

measurements of Ca2+, excitation wavelength alternated between 340 and 380 nm every

second, and fluorescence intensity was monitored at an emission wavelength of 510 nm.

For measurement of Sr2+ and Ba2+, excitation wavelengths of 350 and 390 nm were used.

Intracellular free divalent cation concentrations were calculated as previously described

2+ 2+ (159) using kd values of 224 nM, 780 nM, and 2.62 µM for fura-2 binding to Ca , Ba , and Sr2+, respectively. All measurements were performed at 37oC. For measurement of

2+ 2+ 2+ [Ca ]i or [Ba ]i in the presence of a Ca chelator, cells were first harvested and

incubated in HBS containing 50 µM BAPTA/AM for 40 min at 37oC. Following

centrifugation, the cells were resuspended in HBS containing Fura-2/AM and treated as

described above.

Measurement of vital dye uptake. An aliquot (2 ml) of dispersed cells suspended in

HBS at 37oC was placed in a cuvette. Following addition of ethidium bromide (EB; final

concentration of 2.5 µM), fluorescence was recorded at 1 second intervals as a function of time with excitation and emission wavelengths of 302 and 590 nm, respectively. EB fluorescence values were corrected for background (extracellular) dye fluorescence and expressed as a percentage relative to the value obtained following complete permeabilization of the cells with 50 µM digitonin. Uptake of propidium iodide (PrI;

81 final concentration of 2 µM) was determined as described for EB with excitation and emission wavelengths of 536 and 617 nm, respectively. MTX-induced COP activity was estimated from the maximum slope of the change in EB fluorescence as a function of time after MTX. EB influx from individual fluorescence recordings, was determined before MTX-induced cell lysis and therefore reflects an accurate estimate of COP activity under each condition examined. Time to cell lysis was defined and quantified as the time to 50% PrI uptake.

Transfection of BAECs with GFP constructs. Cells were seeded onto 35 mm culture dishes, and maintained until they reached 90-95% confluence. A single dish of cells was transfected with 2 µg of pEGFP-C1 cDNA as previously described (47), using

Lipofectamine 2000 (Invitrogen). Twenty-four hr following transfection, the cells were dispersed with trypsin/EDTA, and reseeded onto 12 mm glass cover slips (6 – 9 cover slips per 35 mm dish).

Time-Lapse Videomicroscopy. BAECs in complete-DMEM were sparsely seeded on circular glass cover slips and used within 1-3 days of seeding. The cover slips were mounted in a temperature-controlled perfusion chamber and placed on the stage of a

Leica DMIRE2 inverted microscope. The cells were illuminated with light from a 175 watt xenon lamp using filter cubes appropriate for EB and PrI (Leica N21) or GFP (Leica

L5). Epifluorescence was recorded using a SPOT camera (Diagnostic Instruments,

Sterling Heights, MI) and images were acquired and analyzed using SimplePCI imaging software (Compix Inc., Cranberry Township, PA). During each experiment, phase and

dual fluorescence images were sequentially collected at 30 sec intervals with shutter

controllers switching between light and fluorescent illumination. The fluorescence

images were used to quantify dye uptake or GFP loss. For dye uptake, a region over the

nucleus of individual cells was defined and the average fluorescence intensity of the

region was quantified as a function of time. Phase images were digitally merged with the

corresponding fluorescent images and time-lapse videos were created using the

SimplePCI software. To quantify total GFP fluorescence, a region of interest was drawn

around the GFP-positive cell such that it fully enclosed the cell throughout the entire

experiment (i.e. including any membrane blebs). The corrected total GFP signal was

obtained by subtracting the average background level determined from a nearby control

region lacking cells, over the entire region of interest. The background-subtracted GFP

fluorescence was summed for all the pixels within the region of interest. This data was

then normalized to the baseline established during the first five min to enable comparison

between cells.

Statistical treatment of the data. All experiments were performed at least three times

and in most instances, the experiments shown in each panel of a single figure are paired

(i.e. performed on the same day on the same batch of cells). For cuvette-based

experiments, fluorescence was collected at 1 sec intervals and the curves shown in each

figures are the mean values from at least three independent experiments. Unless

otherwise indicated, the symbols represent means ± SE values that, for clarity, are only shown at selected time points. Where indicated, mean values were compared using the paired Student t-test with p < 0.05 considered significant. For single cell measurements,

83 fluorescence values were determined as described above and are plotted for each cell in the field of view as a different color. Time-dependent changes in cell morphology are shown as a montage of selected images; representative videos corresponding to the indicated experiment are available in the Supplemental Material (Video1.avi,

Video2.avi, Video3.avi, and Video4.avi).

RESULTS

MTX-induced cell death cascade. MTX initiates sequential changes in

plasmalemmal permeability that culminate in oncotic cell death (45,47). As seen in Fig

3.1A, addition of MTX to a population of endothelial cells suspended in a cuvette, causes

2+ a concentration-dependent increase in [Ca ]i as indicated by the change in fura-2

2+ fluorescence ratio. The initial rise in [Ca ]i, which reflects activation of CaNSC, is followed closely in time by the biphasic uptake of ethidium bromide (EB; Fig 3.1B). The first phase of EB uptake reflects the opening of large pores in the plasmalemma (COP) and is directly proportional to the MTX concentration (Fig 3.1B). The second phase of

MTX-induced EB uptake is known to be associated with the release of heterologously expressed green fluorescence protein (GFP; MW=27 kDa) and with the release of the endogenous enzyme, lactate dehydrogenase (LDH; MW=140 kDa) (45,47). Thus, the second phase of EB uptake is indicative of cell lysis. Recent studies suggest that propidium iodide (PrI) may exhibit low permeability via COP, and that uptake of PrI may be a good index of cell lysis (47). To test this hypothesis, the MTX-induced uptake of

PrI was examined. As seen in Fig 3.1C, the rapid uptake of PrI correlated in time with the second phase of EB uptake, confirming that PrI uptake reflects the lytic phase. It should be noted that following cell lysis, fura-2 is released from the cell to the extracellular solution. Thus, at early times after MTX addition, the fura-2 signal

2+ accurately reflects [Ca ]i, whereas at later times (i.e., during the lytic event) the

fluorescence ratio reflects both intracellular and extracellular fura-2. For this reason,

2+ calibrated [Ca ]i values are only shown for the period of time prior to cell lysis (Fig

3.1A).

Previous studies in BAECs have shown that the MTX-induced uptake of EB is

2+ 2+ blocked by pharmacological inhibition of Ca influx (46) suggesting that a rise in [Ca ]i is necessary for downstream events in the cell death cascade. To determine if a rise in

2+ 2+ [Ca ]i is necessary or sufficient, we examined the effect of the Ca ionophore,

ionomycin, on the uptake of EB (Fig 3.2). Addition of ionomycin (20 µM) to BAECs

2+ produced an increase in [Ca ]i and a biphasic uptake of EB virtually identical to that

2+ observed with MTX. These results suggest that a rise in [Ca ]i is sufficient to initiate the cell death cascade.

Divalent cation selectivity. It is well known that Sr2+ will substitute for Ca2+ in a number of cellular reactions, but that Ba2+ is a poor surrogate for Ca2+. In particular, the

involvement of high affinity Ca2+ binding proteins can be revealed by evaluation of the

divalent cation potency sequence (61) (Table 1). To determine divalent cation selectivity

of each step in the cell death cascade, fura-2 fluorescence, EB uptake, and PrI uptake

were measured in parallel following the addition of MTX to endothelial cells suspended

in solutions in which Ca2+ was replaced by either Sr2+ or Ba2+. As seen in Fig 3.3A, 1

min after addition of 0.3 nM MTX, the cytosolic Sr2+, Ca2+, and Ba2+ was 9850 ± 105,

803 ± 139, and 452 ± 60 nM, respectively. Within 3 min of MTX addition however,

2+ 2+ 2+ [Ca ]i and [Ba ]i were essentially the same whereas [Sr ]i remained ~10-fold higher.

(At times longer than ~4 min, fura-2 approaches saturation and therefore, the values do

not accurately reflect concentration.) Importantly, these results demonstrate that Ca2+,

Sr2+, and Ba2+ enter the cell in response to MTX, and that the presence of the surrogate

cations in the extracellular solution does not prevent activation of the CaNSC.

To determine the effect of divalent cation substitution on activation of COP and

cell lysis, the uptake of EB was examined (Fig 3.3B). The EB uptake profile in the

presence of Sr2+ was remarkably similar to that observed with Ca2+. There was no

detectable difference in the first phase of EB uptake (i.e., activation of COP) and on

average, only a small delay in time to cell lysis was observed when Ca2+ was replaced by

Sr2+. When Ca2+ was replaced by Ba2+, the first phase of EB uptake was slightly delayed, and COP activity, as indicated by the rate of EB uptake (i.e., the slope), was slightly reduced (7.2 ± 0.5 versus 5.9 ± 0.9 % per min, n=11, p<0.001). The major effect of replacing Ca2+ with Ba2+ however, was the apparent lack of cell lysis, i.e., no second

phase of EB uptake was observed over the time course of this experiment (Fig 3.3B). To determine if in fact the cells ultimately lyse in Ba2+-containing solutions, PrI uptake was

measured over a longer time course (Fig 3.3C). As expected from the EB uptake

profiles, PrI uptake was only slightly delayed when Ca2+ was replaced by Sr2+. However, in the presence of Ba2+, PrI uptake was significantly delayed by ~13 min confirming that

cell survival is prolonged by substitution of Ca2+ with Ba2+. It is important to note that

basal uptake of EB and PrI in the absence of MTX was ~6% after 60 min (data not

shown). Thus, the uptake of EB and PrI in the presence of Ca2+, Sr2+, or Ba2+ is

absolutely dependent on prior stimulation with MTX and does not reflect deleterious

effects of long-term incubation of the cells with these Ca2+ surrogates.

To better evaluate the relative selectivity of each phase of the death cascade for

Ca2+ versus Sr2+, the extracellular divalent cation concentrations were varied so that the

2+ 2+ MTX-induced increase in [Ca ]i and [Sr ]i were essentially the same (Fig 3.4A). Under these conditions, both the first and second phase of EB uptake were slightly delayed in

Sr2+ relative to that observed in Ca2+-containing buffer (Fig 3.4B). This result suggests

that Sr2+ has a potency that is less than Ca2+, but greater than Ba2+.

To determine if the effects of Ba2+ on MTX-induced cell death cascade reflect an

alteration in MTX binding and to further explore the effects of divalent cation

substitution, changes in plasmalemmal permeability were examined at concentrations of

MTX (0.05 to 1.0 nM) both above and below the apparent EC50 (determined previously

to be ~0.3 nM). The full data set is shown in Fig 3.5 and the summary dose-response curves are shown in Fig 3.6. The activity of CaNSC was estimated, in a pair-wise

2+ 2+ fashion, from the initial rate of change in [Ca ]i or [Ba ]i determined before activation

of COP. As seen in Fig 3.5A&D and 3.6A, MTX-induced divalent cation influx, was

little affected by replacing Ca2+ with Ba2+, i.e., there is little or no shift in MTX

concentration-response curve. We next examined COP activity in Ca2+ versus Ba2+-

containing solutions estimated from the maximum slope of EB uptake before cell lysis

(Fig 3.5 B&E and 3.6B). At 0.05 and 0.1 nM MTX, COP activity was on average 1.8-

and 1.4-fold greater in Ca2+ versus Ba2+, respectively. At 0.3 nM MTX, the difference was only 1.1-fold, and at 1.0 nM MTX COP activity in Ca2+ was slightly reduced to 0.82

of that seen in the presence of Ba2+, i.e., the Ca2+ curve appears to cross-over the Ba2+ curve. A similar cross-over phenomenon was previously observed for EB uptake in

THP-1 monocytes when stimulated by high concentrations of MTX (161), suggesting that

2+ a rapid and large increase in [Ca ]i produced by high MTX causes feedback inhibition of

COP activity. These results suggest that there may in fact be an change in COP activity

in Ba2+ versus Ca2+-containing solutions, but the MTX concentration required is similar

for both divalent cations tested. We next examined in a pair-wise fashion, the time to cell

lysis as indicated by PrI uptake (Fig 3.5 C&F and 3.6C). Clearly, the time to cell lysis

was significantly longer at each concentration of MTX examined when Ca2+ was replaced

by Ba2+. These results provide further support for the conclusion that Ba2+ has little

effect on MTX binding or its ability to activate CaNSC, but that some intracellular steps

involved in COP activation and the kinetics of cell lysis are extremely sensitive to

replacement of Ca2+ with Ba2+. If in fact, the effect of Ba2+ on the cell death cascade

reflects interaction with intracellular proteins, we reasoned that simply chelating

cytosolic divalent cation should produce a similar delay in activation of COP and delay in

cell lysis. To test this hypothesis, the effect of a low concentration of MTX was

examined in cells loaded with the Ca2+ chelator, BAPTA. As seen in Fig 3.7A and 3.7B,

2+ loading of the cells with BAPTA significantly delayed the MTX-induced rise in [Ca ]i

2+ and delayed both phases of EB uptake, again suggesting that a rise in [Ca ]i is necessary

2+ for both COP activation and cell lysis. The MTX-induced increase in [Ba ]i and EB

uptake via COP were likewise, significantly delayed (Fig 3.7C and 3.7D). These results provide additional evidence that activation of COP is sensitive to intracellular Ca2+ and

that the delay observed in Ba2+-containing solutions likewise reflects an intracellular

event.

Although all three divalent cations tested support MTX-induced cell death, the results suggest that the proteins and/or enzymes involved in activation of COP and subsequent cell lysis exhibit either a lower affinity for Ba2+ versus Ca2+ or Sr2+, or a lower efficacy with Ba2+ bound. Alternatively, Ba2+ may actually inhibit various steps in the cell death cascade. To distinguish between these possibilities, low concentrations of

Ca2+ were re-admitted to the Ba2+-containing extracellular buffer and the response to

MTX examined (Fig 3.8). Re-addition of 0.1 to 0.5 mM Ca2+ to the Ba2+-containing buffer produced a graded leftward shift in the first phase of EB uptake (Fig 3.8A and A′) and in the time to cell lysis as indicated by the leftward shift in both the second phase of

EB uptake and PrI uptake (Fig 3.8B). These results are consistent with the conclusion that Ba2+ does not inhibit the cell death cascade, and that the affinity of the various steps for Ca2+ is relatively high and much greater than that for Ba2+.

Effect of divalent cation substitution on cell morphology. Previous experiments revealed that BAECs challenged with MTX exhibit dramatic changes in cell morphology (45,47).

To determine the effect of Ba2+ substitution, each phase of the MTX-induced cell death cascade was compared to time-dependent changes in cell morphology using simultaneous phase and fluorescence videomicroscopy. For these studies, BAECs were transfected with a GFP construct and seeded onto glass coverslips. The cells were bathed in a either

Ca2+- or Ba2+-containing solution along with the vital dye, EB. The first phase of EB uptake at the single cell level was used as an index of COP activity. The second phase of

EB uptake and the loss of GFP were used as indexes of single cell lysis. In parallel experiments, the loss of GFP and the uptake of PrI were simultaneously monitored as a

90 third index of cell lysis. Phase and fluorescence image pairs were recorded every 30 sec and quantified at the single cell level as described in Materials and Methods. The representative montage in Fig 3.9 shows phase, GFP, EB, and merged images of an endothelial cell before, and at various times after, the addition of MTX. As previously reported (45), addition of MTX (at time 5 min) caused the formation of membrane blebs that develop within 5-10 min of MTX addition and appear as balloon-like structures on the surface of the cells (Fig 3.9A). Formation of membrane blebs occurred during the first phase of EB uptake (Fig 3.9B) and therefore correlates with activation of COP. At time 18 min, GFP begins to leave the cell and by 23 min cell-associated green fluorescence is non-detectable. The second phase of EB uptake correlates in time with the loss of GFP (Fig 3.9B). The EB uptake profile was the same whether quantified in a region within the nucleus or within the cytoplasm (Fig 3.9C) confirming that the biphasic profile observed does not reflect limited access of the dye to the nucleus. Note from the images in Fig 3.9A, that blebs do not rupture during and after cell lysis, but rather continue to appear as smooth spherical structures without obvious tears or discontinuities. In fact, during and after the loss of GFP, the blebs continue to grow in size. This is most evident in time-lapse video (Video1.avi and Video2.avi) that accompany this manuscript. The same experiment was repeated using solutions in which the Ca2+ was replaced by Ba2+. A representative montage with three cells in the field of view is shown in Fig 3.10. All of the cells exhibit the first phase of EB uptake indicative of COP, but the time to activation of COP is delayed relative to that observed in Ca2+- containing solution. Over the time frame shown, only one of the green cells exhibits the second phase of EB uptake and a rapid loss of GFP. Furthermore, the time to lysis for the

cell shown was 40 min after MTX addition, much longer than the time observed in Ca2+-

containing solutions. Thus, as suggested by the cuvette studies, Ba2+ 1) delays activation

of COP, 2) has relatively little effect on MTX-induced COP activity once activated, and

3) greatly delays cell lysis. Importantly, blebbing is either not observed or greatly

attenuated when the cells are challenged with MTX in Ba2+-containing solution. In particular, when Ba2+ replaces Ca2+ the cells tended to exhibit a more general

enlargement or swelling with little or no bleb formation. Furthermore, if blebs do form in

the presence of Ba2+, they generally lack a defined neck region needed to give the distinct

spherical appearance typical of the dilated blebs seen in Ca2+-containing solutions. Many

times the cells fail to bleb at all or exhibit only a single bleb in the presence of Ba2+

(Video3.avi and Video4.avi).

The effect of MTX in either Ca2+- or Ba2+-containing solution was examined on

several coverslips and the loss of GFP was correlated with either EB uptake or PI uptake.

The individual response for each cell examined is shown in Fig 3.11 and the mean ± SD values are summarized in Fig 3.12. In Ca2+-containing buffer, the uptake of EB was

biphasic at the single cell level. Additionally, the rapid loss of GFP correlated closely in

time with both the second phase of EB uptake and the rapid phase of PI uptake, i.e.,

between 15-25 minutes the majority of cells release GFP and rapidly stain with both EB

and PI (Fig 3.11B and 3.11C). Thus, MTX-induced EB and PI uptake observed in the

population-based cuvette assays were qualitatively recapitulated at the single cell level.

In Ba2+-containing solution it is clear that MTX-induced activation of COP is delayed as

indicated by the time lag before the first phase of EB uptake (Fig 3.12B). Furthermore,

once activated, COP activity (i.e., the slope of the first phase of EB uptake), is the same

in both Ca2+ and Ba2+-containing solutions (Fig 3.12B). However, both the second phase

of EB uptake and the rapid phase of PI uptake are significantly delayed. Furthermore,

there is substantial cell-to-cell variability in the time to cell lysis in Ba2+. In Ca2+, the majority of cells lyse within a narrow 10-15 min window, whereas in Ba2+, lysis occurs

between 30-60 min and a number of cells (26 of 37) fail to lyse over the time course of

these experiments (Fig 3.12C and 3.12F). Thus, there appears to be substantial

heterogeneity in time to cell lysis when Ba2+ replaces Ca2+. In Ba2+ solution, only 6 of 15

cells challenged with MTX showed a rapid phase of GFP release (Fig 3.12D). The

majority of cells exhibited only a slow decline in GFP fluorescence over the time course

of these experiments. However, the rapid loss of GFP in the 6 cells correlated in time

with the second phase of EB uptake. The slow rundown of GFP fluorescence in these

long time course studies may reflect either photobleaching or more likely, the movement

of GFP out of the focal plane as the cells swell and enlarge in response to MTX.

DISCUSSION

Stimulation of endothelial cells with MTX leads to a well-defined sequence of

permeability changes that culminate in oncotic cell death. First, MTX causes a large

2+ increase in [Ca ]i via the activation of CaNSC. Second, there is an opening of large

endogenous pores (i.e. COP) that allow for the uptake of EB. Finally, the cells undergo

lysis, an all or nothing event evidenced by the release of large macromolecules from the

cell and the rapid uptake of PrI. During each phase of the response there is a dramatic

change in cellular morphology characterized by the appearance of membrane protrusions

or blebs that form concurrently with the activation of COP and dilate as the cell

progresses through the lytic phase. As the concentration of MTX increases, there is an

2+ increase in [Ca ]i, an increase in COP activity, and a decrease in the time to cell lysis.

2+ Although these events appear to be sequential and to depend on the rise in [Ca ]i, the

2+ biochemical mechanisms linking each step in the cascade and the specific role of [Ca ]i in each phase of the response remains unknown. Similar changes in plasmalemmal permeability have been observed following stimulation of purinergic receptors of the

P2X7 subtype (161) suggesting commonality in the cell death cascades.

In order to evaluate the role of Ca2+ as either a trigger or modulator of COP

activity and/or cell lysis, we took advantage of the well-known difference in affinity of

various Ca2+ binding proteins for Ba2+ (61). Although Ba2+ and Sr2+ are permeable

through many types of voltage-gated and receptor-operated cation channels, Ba2+ is a

poor substrate for SERCA and PMCA pumps (53,166,176,203), and in general has much

lower affinity for Ca2+ binding proteins of the EF-hand and C2-type when compared with

Ca2+ (61). The results of the present study show that the kinetics and magnitude of each

step in the cell death cascade were very similar when Ca2+ was isosmotically replaced by

Sr2+, with only a slight delay in the average time to cell lysis. A delay in the time to COP activation was revealed in Sr2+ however, when the extracellular divalent cation

concentrations were adjusted to give comparable increases in cytosolic concentrations.

When Ca2+ was isosmotically replaced by Ba2+, COP activation was again slightly delayed and the time to cell lysis was greatly prolonged. These changes noted in Ba2+-

containing solution were observed at both the population and single cell level. Thus,

proteins that couple channel activation with downstream events observed in the cell death

cascade appears to have a lower affinity for Ba2+. Furthermore, in the presence of Ba2+, addition of 0.1 to 0.5 mM Ca2+ to the extracellular buffer produced a concentration-

dependent decrease in the time to COP activation and cell lysis. Overall, these result

provides strong support for the conclusion that proteins with high affinity Ca2+ binding

sites are critically involved in the sequential changes in plasmalemmal permeability that

accompany MTX-induced cell death.

The kinetics of COP formation or activation were delayed by replacement of Ca2+ with either Sr2+ or Ba2+, or by chelation of intracellular Ca2+ or Ba2+ by BAPTA.

2+ Furthermore, simply increasing [Ca ]i by addition of ionomycin produced a bi-phasic

uptake of EB similar to that seen with MTX. These results suggests that an intracellular

step between activation of the CaNSC and COP is affected by Ca2+. The actual

mechanism by which MTX activates COP is unknown. Likewise, the mechanism by

which activation of P2X7 purinergic receptors is linked to formation of COP is unclear. It was originally proposed that P2X7 receptors may aggregate to form larger and larger pore

structures (131,186). However, studies using GFP-tagged receptors have shown that EB

uptake is not associated with changes in receptor distribution or changes in receptor

density (171). Additionally, we have shown that although the permeability of COP is

inversely proportional to dye molecular weight, MTX-induced dye uptake is linear during

the COP phase which is inconsistent with a dilation model (45). The MTX-activated

channel and the channel formed by P2X7 receptor protein are clearly distinct, but the

COP activated by both MTX and purinergic agonists are virtually indistinguishable (161).

This has led to speculation that COP is a separate membrane-associated pore structure that can be activated by a variety of Ca2+-permeable cation channels. In this regard we

have shown that over-expression of P2X7 receptors produces a shift in the MTX

concentration-response curve such that higher concentrations of the toxin are required to

activate COP (161). This result suggests that the MTX channel and the purinergic

channels complete for a common pool of COP and that the channels may be physically

associated with the COP structure. Indeed, rapid pharmacological inhibition of MTX-

induced Ca2+ influx during the first phase of EB uptake, produces an immediate blockade of COP activity, consistent with tight functional coupling between CaNSC activity and

2+ COP (45). A rise in [Ca ]i may therefore be necessary for physical interaction or

coupling between the CaNSC and COP.

The single cell experiments revealed additional differences in the cell death

cascade in the presence of Ba2+. Firstly, Ba2+ caused a desynchronization of cell lysis as

indicated by the dispersion in PrI uptake. In the presence of Ca2+, all of the cells rapidly accumulate PrI within a narrow 10 min window, whereas in Ba2+-containing solution, cell

lysis was delayed and occurred over 35-60 min time span. This heterogeneity with

respect to cell lysis was also evident in the time to the second, rapid phase of EB uptake.

The biochemical reason for the difference in cell synchronization between Ca2+ and Ba2+ is unclear. One possibility is that a critical cellular factor must be exhausted before the cell will lyse and that the degradation or metabolism of this factor is slowed in Ba2+ relative to Ca2+. Since Ba2+ is a poor substrate for Ca2+ pumps and will not be

sequestered within the endoplasmic reticulum, a possible candidate for this protective

factor is ATP. During Ca2+ overload conditions, Ca2+ pumps are expected to be operating

near maximum, consuming large amounts of ATP at a time when mitochondrial function

is probably seriously compromised. However, under Ba2+ overload conditions, pump

activity and hence ATP consumption will be reduced. The hypothesis that MTX-induced

cell lysis occurs when ATP is reduced to a critical level is currently under investigation.

Secondly, the single cell experiments showed that Ba2+ substitution dramatically

altered the membrane blebbing profile. In Ca2+-containing solution, blebs initially form

during and following COP activation. Once formed, blebs remain relatively constant in

size until the lytic event at which time a dramatic bleb dilation is observed. Interestingly,

the cell morphology remains relatively intact even during the massive bleb dilation,

consistent with a previous suggestion that membrane blebbing may be a mechanism by

which the cell regulates or relieves large increases in intracellular pressure(5). The blebs

seen in Ca2+ solutions also have a narrow neck at the point of attachment to the cell,

which contributes to the balloon-like appearance. However, the MTX-induced bleb

formation is dramatically attenuated when Ca2+ is replaced by Ba2+ and a more

generalized cell swelling is often observed. If blebs do form in the presence of Ba2+, they lack a defined neck structure consistent with the hypothesis that a protein with high affinity for Ca2+ is needed for maintaining the integrity of the neck complex. Although the mechanisms responsible for necrotic and apoptotic membrane blebbing appear to be different (5), previous studies on membrane blebbing have shown that the actin-myosin cytoskeleton forms a ring centered at the neck of membrane blebs and that myosin light chain phosphorylation via myosin light chain kinase (MLCK) plays an important role in bleb formation (5,117,193). MLCK is a Ca2+-calmodulin dependent enzyme and is

therefore expected to have a low affinity for Ba2+.

In conclusion, the results of the present study show for the first time that high affinity

Ca2+-binding proteins are involved in regulating changes in plasmalemmal permeability

in response to MTX. Specifically, high affinity Ca2+ binding proteins appear to play a critical role in the transition from CaNSC to COP and in the transition from COP to lytic pore. Although Ca2+-sensitive phosphatases (eg. calcineurin), proteases (eg. calpain),

2+ and/or lipases (eg. PLA2) may be indirectly involved, a more direct effect of Ca on the

formation of COP and the assembly or activation of a lytic death channel cannot be

excluded. Additional studies will be necessary to determine the identity of the Ca2+ binding proteins involved in each step and to understand their mechanism of action at the plasma membrane.

Table 1. Divalent cation selectivity of known Ca2+ binding domains Ca2+ Binding domain Apparent divalent cation REF. affinity

Ca2+ Sr2+ Ba2+ (µM) (µM) (µM) EF hand Phosphodiesterase via calmodulin 2.5 25 > 2500 (21) MLCK via calmodulin 0.33 85 39000 (138) Adenylyl Cyclase VIII 0.3 12 500 (61) Skeletal muscle contraction 0.89 26 361 (177) Skeletal muscle contraction 0.80 26 700 (59) Galactose binding protein 1.4 190 12000 (37) Phospholipid scramblase 28.7 308 2783 (178) Nitric oxide synthase 0.20 N.D. 50 (218)

C2 domain Synaptotagmin I 5.4 177 254 (99) cPLA2 2 43 >2000 (125) PKC 10 200 > 1000 (158)

Other Na+-Ca2+ exchanger 0.3 N.D 10 (195)

Rank order of potency EF hand Calcineurin Sr2+ > Ca2+ > Ba2+ (214)

C2 domain Synaptotagmin I Ca2+ > Sr2+ > Ba2+ (99) Synaptotagmin III Ca2+ > Sr2+ ≥ Ba2+ (51) Phospholipase C Ca2+ > Sr2+; Ba2+(N.E.) (3) PI-3-kinase Ca2+ >> Sr2+; Ba2+(N.E.) (3)

Other Na+-Ca2+ exchanger Ca2+ > Sr2+ >>Ba2+ (190) Platelet aggregation Ca2+ > Sr2+ > Ba2+ (11) cAMP affinity of CNG channels Ca2+ > Sr2+ >>Ba2+ (106) Ca2+-ATPase Ca2+ > Sr2+ >>Ba2+ (56,198) N.D. not determined; N.E. no effect

FIGURES

Fig 3.1. Effect of MTX on plasmalemmal permeability. Panel A: Fura-2-loaded bovine

aortic endothelial cells (BAECs) were suspended in HBS and the fluorescence ratio was

recorded as a function of time as described in Material and Methods. Two traces are

shown superimposed. MTX, (0.3 nM (z) or 0.05 nM ({)), was added to the cuvette at

2+ the time indicated by the arrow. Main panel shows calibrated values for [Ca ]i at times before cell lysis. Inset shows the ratio values over a longer time course. In this and all subsequent figure legends, values given indicate final concentrations. Panel B: Ethidium bromide (EB) uptake was determined as described in Material and Methods. Two traces are shown superimposed. For each trace, BAECs were suspended in HBS at 37° C. EB

(2.5 µM) was added to the cuvette at 20 sec and MTX (0.3 nM, z or 0.05 nM, {) was added at 100 sec. Panel C. Propidium iodide (PrI) uptake was determined as described in

Material and Methods. PrI (2 µM) was added to the cuvette at 20 sec and MTX (0.3 nM, z or 0.05 nM, {) was added at 100 sec. Curves shown are representative of at least 3 experiments. The timing of each phase of the response is indicated by the dashed lines.

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2+ Fig 3.2. Effect of ionomycin on the change in [Ca ]i and EB uptake in BAECs.

2+ [Ca ]i (Panel A) and EB uptake (Panel B) were measured in BAECs as described in the

legend to Fig 3.1. Ionomycin (20 µM) was added at the time indicated by the arrow. The

inset in Panel A shows fura-2 ratio values over a longer time course. Curves represent

mean values of 3 independent experiments; symbols represent mean ± SE values at selected time points.

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103

Fig 3.3. Effect of divalent cation substitution on COP activation and cell lysis. Three traces are shown superimposed in each panel. BAEC cells were suspended in HBS containing 1.8 mM Ca2+ ({), Sr2+ (V), or Ba2+ (z). Panel A. Increase in divalent cation concentration was calculated from fura-2 fluorescence ratios after the addition of MTX

(0.3 nM). Uptake of EB (Panel B) and PrI (Panel C) in each buffer was determined from the increase in fluorescence as a function of time. Note that the time frame is different in each panel. Curves represent mean values of 3 independent experiments; symbols represent mean ± SE values at selected time points.

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105

Fig 3.4. Comparison of Ca2+ with Sr2+. Two traces are shown superimposed in each panel. BAEC cells were suspended in HBS containing either 10 mM Ca2+ ({) or 1.25 mM Sr2+ (V). Panel A. Increase in divalent cation concentration was calculated from fura-2 fluorescence ratios after the addition of MTX (0.3 nM). Uptake of EB (Panel B) in each buffer was determined from the increase in fluorescence as a function of time.

Curves represent mean values of 3 independent experiments; symbols represent mean ±

SE values at selected time points.

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Fig 3.5. Effect of Ca2+ replacement by Ba2+ on the MTX concentration-response

2+ 2+ curve. Four traces are shown superimposed. The [Ca ]i, [Ba ]i, EB uptake, and PrI uptake were determined as described in the legend to Fig 3.3 at concentrations of MTX of

0.05 (U), 0.1 (S), 0.3 ({), and 1.0 nM (z). Increase in Ca2+ (Panel A) or Ba2+ (Panel

D) concentration was calculated from fura-2 fluorescence ratios after the addition of

MTX. Uptake of EB (Panel B or E) and PrI (Panel C or F) was determined from the increase in fluorescence as a function of time in Ca2+- or Ba2+-ECS, respectively. Curves represent mean values of 3 independent experiments; symbols represent mean ± SE values at selected time points.

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Fig 3.6. Effect of Ca2+ replacement by Ba2+ on the CaNSC activity, COP activity, and time to cell lysis dose response. Values presented were calculated from the

2+ 2+ individual fluorescence traces in Fig 3.5. The [Ca ]i, [Ba ]i, EB uptake, and PrI uptake were determined as described in the legend to Fig 3.3 at concentrations of MTX of 0.05,

0.1, 0.3, and 1.0 nM in the presence of either Ca2+ ({) or Ba2+ (z). The d[divalent]/dt was determined from the maximum slope of calibrated fura-2 traces before activation of

COP, i.e., before EB uptake. The activity of COP was quantified as the slope of the first phase of EB uptake before cell lysis, i.e., before PrI uptake. The time to cell lysis was quantified as the time to 20% PrI uptake. Values were determined from individual fluorescence traces and the symbols shown represent the mean ± SE values from 3 experiments.

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2+ Fig 3.7. Effect of BAPTA-loading on MTX-induced change in [Ca ]i and

2+ [Ba ]i and the associated EB uptake. Two traces are shown superimposed in each

panel. BAECs were loaded with fura-2 and BAPTA as described in Material and

Methods and suspended in HBS containing either 1.8 mM Ca2+ (Panels A and B) or 1.8

mM Ba2+ (Panels C and D). Panel A and C. The net change in divalent cation concentration was calculated from fura-2 fluorescence ratios after the addition of MTX

(0.05 nM) in control (z) or BAPTA-loaded cells ({). Panel B and D. The uptake of EB was determined following addition of MTX (0.05 nM) in control (z) or BAPTA-loaded cells ({). Curves represent mean values of 3 independent experiments; symbols represent mean ± SE values at selected time points. Note that the y-axis is expanded in

Panel D to highlight the first phase of EB uptake.

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Fig 3.8. Effect of Ca2+ readmission on COP activation and cell lysis. Five traces are shown superimposed in each panel. BAEC cells were suspended in HBS containing either 1.8 mM [Ca2+] ({) or 1.8 mM [Ba2+] (z). Where indicated, Ba2+-ECS was

supplemented with Ca2+ to obtain a final concentration of 0.1 (T), 0.2 (◆), or 0.5 mM

2+ 2+ 2+ 2+ [Ca ]e (). Panel A. EB uptake was recorded in normal Ca -HBS, Ba -HBS, or Ca -

supplemented Ba2+-HBS. Panel A’. Data from (A) was re-plotted on expanded scale to highlight the first phase of EB uptake. Panel B. PI uptake was recorded in normal Ca2+-

HBS, Ba2+-HBS, or Ca2+-supplemented Ba2+-HBS (note the expanded time scale). Curves

represent mean values of 3 independent experiments; symbols represent mean ± SE values

at selected time points.

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Fig 3.9. Simultaneous measurement of MTX-induced GFP loss and EB uptake in single BAECs. BAECs, transfected with GFP, were grown on glass coverslips, mounted on the stage of an inverted fluorescence microscope, and bathed in normal HBS containing EB at 37o C. Sequential phase and dual fluorescent images were recorded every 30 sec for 40 min as described in Material and Methods. MTX (0.3 nM) was added to the bath at t = 5 min. Panel A. each row of the montage shows 4 images from a selected cell (phase, GFP, EB, and merged phase/dual fluorescence) taken at the indicated time points. Size bars = 10 micron. Panel B. GFP (green line) and EB (red line) fluorescence from each image was quantified as described in Material and Methods and is shown as a function of time. Panel C. Simultaneous GFP release (z) and EB uptake as function of time were quantified for 29 individual cells challenged with MTX

(0.3 nM) as described in Panel B. EB uptake was determined as the average pixel intensity in a region over the nucleus (T) or over the cytoplasm ({). Symbols represent mean ± STANDARD DEVIATION values at selected time points. Time-lapse video of this experiment (Video1.avi) is available as part of the supplementary materials.

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Fig 3.10. Effect of Ba2+ on MTX-induced cell death cascade in single BAECs.

The experiment was performed and fluorescence quantified exactly as described in the legend to Fig 3.9 with BAECs bathed in Ba2+-HBS. Time-lapse video of this experiment (Video3.avi) is available as part of the supplementary materials.

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Fig 3.11. Composite single cell fluorescence data. Experiments were performed and the fluorescence quantified as described in Figs 3.10 and 3.11 in the presence of Ca2+-

HBS (Panels A, B, and C) or Ba2+-HBS (Panels D, E, and F). Each line represents a single cell from several (n = 3-5) independent experiments. In all panels MTX (0.3 nM) was added at t = 5 min. The total number of cells evaluated under each condition is shown in parentheses. Note that the loss of GFP fluorescence was simultaneously monitored in parallel experiments with either EB or PrI uptake.

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Fig 3.12. Average single cell responses. The average single cell GFP (Panel A), EB

(Panel B), or PI (Panel C) fluorescence values were calculated from the data shown in

Fig 3.11 in either Ca2+-HBS (z) or Ba2+-HBS ({). Because of the large number of cells examined, the symbols shown at selected time points represent mean ± STANDARD

DEVIATION values.

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Chapter 4

Summary and Future directions

125

SUMMARY - Chapter 2

To evaluate interaction of vanilloid receptors (TRPV1) with endogenous Ca2+ signaling mechanisms, TRPV1 was expressed in Sf9 insect cells using recombinant baculovirus. Stimulation of TRPV1-expressing cells, but not control Sf9 cells, with resiniferatoxin (RTX), capsaicin, or anandamide, produced an increase in cytosolic free

2+ 2+ Ca concentration ([Ca ]i), with EC50 values of 166 pM, 24.5 nM, and 3.89 µM, respectively. In the absence of extracellular Ca2+, both capsaicin and RTX also caused an

2+ increase in [Ca ]i with EC50 values of ~10 µM and 10 nM, respectively. This TRPV1- induced release of Ca2+ from intracellular stores was not blocked by U73122, suggesting that phospholipase C was not involved. Substantial overlap was found between the thapsigargin- and RTX-sensitive internal Ca2+ pools and confocal imaging showed that intracellular TRPV1 immunofluorescence co-localized with the endoplasmic reticulum

(ER) targeting motif, "…KDEL…". To determine if TRPV1-induced mobilization of intracellular Ca2+ activates endogenous store-operated Ca2+ entry (SOCE), the effect of 2-

aminoethoxydiphenyl borate (2-APB) on Ba2+ influx was examined. 2-APB blocked thapsigargin-induced Ba2+ influx, but not RTX-induced Ba2+ entry. In the combined presence of thapsigargin and a store-releasing concentration of RTX, the 2-APB-sensitive component was essentially identical to the thapsigargin-induced component. Similar results were obtained in HEK cells stably expressing TRPV1. These results suggest that

TRPV1 forms agonist-sensitive channels in the ER, that when activated, release Ca2+ from internal stores, but fail to activate endogenous SOCE. Selective activation of intracellular TRPV1, without concomitant involvement of plasmalemmal Ca2+ influx 126 mechanisms, could play an important role in Ca2+ signaling within specific subcellular microdomains.

127

FUTURE DIRECTIONS - Chapter 2

While the present studies provide some insight into the possible function of ion channel

proteins expressed in intracellular membranes, further experimental investigation will be

necessary to determine the exact nature of their influence on cellular physiology.

Physiological role of native TRPV1 in the endoplasmic reticulum.

Previous studies have established that TRPV1 is expressed in intracellular membranes in

dorsal root ganglion neurons (102,135,213). Therefore it will be important to determine

the effect that expression and activation of a nonselective cation channel has on both

endoplasmic reticular and cellular function. TRPV1 mediated release of Ca2+ from internal stores could play a variety of roles in cellular functioning including the modulation of excitability, exocytosis, gene expression, intracellular signaling and cell viability. Thus it will be important to understand (1) which specific intracellular Ca2+ stores are gated by TRPV1; (2) how intracellular TRPV1 is activated and regulated; (3) how TRPV1 gated Ca2+ stores interact both functionally and physically with other organelles and proteins; and (4) how these factors combine to influence cellular functions.

How is intracellular TRPV1 activated and modulated ?

The vanilloid receptor agonists capsaicin and resiniferatoxin are not found

endogenously. In addition, the high temperatures (>42° C) and acidic pH sufficient to

activate TRPV1 are not normally experienced either. Therefore, attempts have been made

to determine the identity of endogenous vanilloid agonists. Plasma membrane TRPV1 has

also been shown to be activated by endogenous lipids including anadamide (133), 12- 128

HPETE (83), and N-arachidonoyl-dopamine (82). The response to these agonistis has

also been shown to be sensitized by phosphorylation (32,134,147,205) and mildly acidic pH (133). This raises the possibility that endogenous agonists or lipids that individually may be sub-threshold for TRPV1 activation may act synergistically with accompanying sensitizing factors like protein kinases to activate TRPV1. While there is growing evidence of endogenous agonists activating TRPV expressed on the plasma membrane, little is known regarding the ability of endogenous lipids like anandamide, 12-HPETE, and N-arachidonoyl-dopamine to induce TRPV1-mediated release of Ca2+ from intracellular stores. As yet the TRPV1-mediated release of intracellular Ca2+ has only

been shown using capsaicin and RTX at concentrations above their respective EC50 values. Thus it is important to examine the ability of these lipids to activate intracellular

TRPV1 in a more physiological context. Additionally, it will also be valuable to determine if activation of receptors coupled to PLC like bradykinin are capable of potentiating the TRPV1 mediated release of Ca2+. One recently discovered tool that will greatly aid these studies is IBTU, a high affinity competitive antagonist of the vanilloid receptor that blocks only the plasma membrane Ca2+ influx and not the TRPV1 mediated release of intracellular Ca2+ stores (194). The selectivity of this inhibitor will enable the

TRPV1-mediated release of Ca2+ from intracellular stores to be separated from Ca2+ entry across the plasma membrane. Therefore further studies are necessary to determine if 1) endogenous lipid activators are capable of causing TRPV1 mediated release of Ca2+ from

intracellular stores; and 2) if this release of stores can be potentiated by by sensitizing

factors like PKA, PKC, and PIP2 hydrolyisis.

129

What cellular organelles does intracellularly expressed TRPV1 interact with ?

While intracellular TRPV1 is known to be functional and to co-localize with ER

markers, little is known about its specific localization the nature of other interacting

proteins. The local environment has been shown to influence the initiation and

propagation of Ca2+ signaling events in a variety of cell types (7,48,115). The spatiotemporal dynamics of intracellular Ca2+ signals are known to be shaped by interactions with mitochondria (39,40), the endoplasmic reticulum (113), and the plasma membrane (12). One possibility could be that intracellularly expressed TRPV1 segregates into signalling microdomains. Recent studies in both nonexcitable cells and neurons have shown different surface receptors are linked to specific intracellular Ca2+ stores (33,167). These differences allow the cell to create signaling diversity and to shape the responses to different stimuli in a more specific fashion. This raises the possibility that intracellular TRPV1 may also play a more defined role in Ca2+ signaling than that of a Ca2+ release mechanism. To test this hypothesis, colocalization studies using

immunofluorescence and confocal microscopy could be performed. Specifically, it will

be important to examine: 1) where TRPV1 expressed on the endoplasmic reticulum is

localized within the cells; and 2) what cellular organelles (e.g. mitochondria, ER, and

2+ golgi), Ca release channels (IP3 and ryanodine receptors), and other proteins (PLC,

ATPases, GPCR, cytoskeletal elements, etc.) TRPV1 colocalizes with. A further understanding of the of potential proteins and organelles that could modulate or regulate the TRPV1-mediated release of Ca2+ could provide clues to the role that it plays in

cellular function.

130

How does TRPV1-mediated release of Ca2+ from internal stores affect cellular function ?

Recently it has been reported that menthol activation of TRPM8 in DRG neurons releases Ca2+ from internal stores. This release then facilitates presynaptic glutamate

release and influences the frequency of minature excitatory post synaptic potentials

(mEPSP) (197). It would be valuable to test the hypotheisis that TRPV1-mediated release

of internal Ca2+ stores influences neurotransmitter release and alters synaptic

transmission in a manner similar to TRPM8. A neuronal co-culture model like that

utilized in the menthol study could be used to test this hypothesis. This model is

composed of both DRG neurons and dorsal horn neurons in the spinal cord and has been

used to study synaptic transmission and modulation at the first sensory synapse (62,197).

This would enable the examination of the effect of TRPV1-mediated release of

intracellular Ca2+ stores on several aspects of sensory transmission. Specifically, it may allow for an examination of : 1) the affect on the ionic currents and action potential firing pattern of the presynaptic neuron, 2) the release of neurotransmitters by the presynaptic neuron following Ca2+ store release, and 3) changes in frequency of mEPSP at the post-

synaptic neuron. These results would help to elucidate the role that TRPV1-mediated

release of internal Ca2+ plays in neuronal excitability and synaptic transmission in the

pain pathway.

Role of TRPV1 and the subsequent Ca2+ influx on necrotic or apoptotic cell death.

Activation of TRPV1 has also been shown to cause necrotic cell death both in

vitro and in vivo (19,22,72,135). As discussed in the introduction (Chapter 1) it is 131

thought that activation of Ca2+-permeable nonselective cation channels can activate a common cell death cascade (161). Thus, it would be interesting to investigate the role that

activation of TRPV1 and the subsequent rise in Ca2+ play in the initiation of necrotic/oncotic cell death. The hypothesis to be tested would be that activation of

2+ TRPV1 and the accompanying increase in [Ca ]i lead to a cell death cascade mechanistically similar to that observed with MTX and/or P2X7 stimulation. Specifically,

2+ the effect that activation of TRPV1 and the accompanying increase in [Ca ]i has upon

COP activity, membrane blebbing and cell lysis could be examined at both the population and single cell level. Cells heterologously expressing TRPV1 could be examined in the same manner as MTX treated BAEC cells described in chapter 3. The expected result

2+ would be that following activation of TRPV1 and the accompanying rise in [Ca ]i would cause COP activation, membrane blebbing and cell lysis similar to that observed in

MTX- stimulated cells. This observation would strengthen the argument that there is a ubiquitous, biochemically conserved, cell death cascade that activates in response to Ca2+ overload. TRPV1 is an excellent candidate for this type of study because of its known involvement in cell death in vivo, as well as the multiple synergistic stimuli capable of activating the channel. In addition, the ability of TRPV1 to mediate the release of Ca2+ from internal stores could provide an additional means to elevate cytosolic Ca2+, a key

event in the initiation of necrotic cell death.

132

SUMMARY - Chapter 3

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial

cells (BAECs), a model for Ca2+-overload-induced toxicity, reflects three sequential

changes in plasmalemmal permeability. MTX initially activates Ca2+-permeable, non-

selective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca2+

2+ concentration ([Ca ]i). This is followed by opening of large endogenous cytolytic oncotic pores (COP) that allow molecules <800 Da to enter the cell. Lastly, the cells lyse, not by rupture of the plasmalemma, but rather through activation of a ‘death’ channel that lets large endogenous cytoplasmic proteins like lactate dehydrogenase (140 kDa) leave the cell. These changes in permeability are accompanied by formation of membrane blebs. In this study, we took advantage of the well-known difference in affinities of various Ca2+-binding proteins for Ca2+ and Sr2+ versus Ba2+ to probe their involvement in each phase of the cell death cascade. Using fluorescence techniques at the cell population level (cuvette-based) and at the single cell level (time-lapse videomicroscopy) we found that replacement of Ca2+ with either Sr2+ or Ba2+ delayed both MTX-induced activation of COP, as indicated by the uptake of ethidium bromide, and on subsequent cell lysis as indicated by the uptake of propidium iodide or the release of cell-associated GFP. MTX-induced responses were mimicked by ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single cell level revealed that Ba2+ not only delayed the time to cell lysis, but also caused a desynchronization of the lytic phase. Lastly, membrane blebs, which in Ca2+-containing solutions were numerous and spherical, were poorly defined and greatly reduced in 133

number in the presence of Ba2+. Taken together, these results suggest that intracellular high affinity Ca2+ binding proteins are involved in the MTX-induced changes in plasmalemmal permeability responsible for cell demise.

134

FUTURE DIRECTIONS - Chapter 3

Determine the identity the MTX activated channel in BAEC cells.

2+ Activation of nonselective cation channels and subsequent increase in [Ca ]i following stimulation with MTX has been observed in all cells tested to date

(10,35,44,64,94,123,160). However, the exact identity of the channel(s) activated by

MTX remain unknown. Therefore it will be valuable to determine the identity of the

MTX activated channel. One potential option to determine the identity of the MTX- activated channel would be to attempt to clone the channel gene. In this respect, expression cloning is often used as strategy to clone genes known to be activated or up- regulated in the response to specific stimuli. This approach has been used to clone genes for other ligand-gated nonselective cation channels including TRPV1(19) and TRPM8

(114). There are several obstacles in attempting to use this approach for the MTX- stimulated channel. Primarily, the ubiquitous nature of the MTX-activated channel makes it difficult to find an expression system that does not have endogenous MTX-activated channels. Potential candidate expression systems used in expression cloning like Xenopus oocytes have been shown to express endogenous MTX-activated channels (10). Therefore it is likely that MTX stimulation could activate not only an expressed channel but also endogenous channels as well. A second possible option would be to determine which protein(s) MTX acts as a high affinity agonist of using MTX binding studies. This is also difficult because MTX is a organic product purified from a dinoflagellate and obtaining radiolabeled MTX for binding studies is not presently possible. A third option would be to attempt to biochemically purify the MTX-stimulated channel using MTX itself. 135

However, this approach is severely limited for similar reasons to the binding study.

Maitotoxin does not contain functional groups amenable to chemical crosslinking and

thus could not be utilized for biochemical purification in an affinity column. In addition

MTX, being a natural product is not available in large quantities, and is also highly toxic.

Determining the identity of the MTX-activated channel may be possible with

more indirect approaches. This would involve identifying known Ca2+-permeable

nonselective cation channels with biophysical properties similar to MTX-activated

channels. An approach like this would involved direct electrophysiological comparisons

of the both the candidate CaNSC and MTX-activated channels. This would allow for

direct evaluation of biophysical properties examined at both the single channel and whole

cell recording configuration. Once candidate MTX-activated channels are identified,

techniques such as small interference RNA (29,42) could be utilized to knock down the

MTX response. This technique has been used with some success to evaluate the

contribution of TRPM7 to Ca2+ entry prior to and during anoxic neuronal cell death (1).

This approach does operate under the assumption that MTX is activating a single channel

type and not multiple channels in a cell.

One potential candidate for the MTX-stimulated channel could be the bifunctional channel-kinase, TRPM7. TRPM7 is a member of the melastatin family of Ca2+- permeable nonselective cation channels, and is unique due to its dual function as both a ion channel and Ser/Thr protein kinase (156). TRPM7 has several distinguishing characteristics including a ubiquitous tissue distribution, nonspecific blockade by the

PLC inhibitor U73122 and its inactive analogue U73343, and blockade by intracellular

Mg2+ (124,157). TRPM7 has also been shown to play a key role in Ca2+ entry during 136

anoxic neuronal cell death (1). Like TRPM7, the MTX activated channel is ubiquitous

and has been observed in all cell types examined to date (161). There is also evidence

2+ that MTX-induced rise in [Ca ]i is blocked in a PLC-independent manner by U73122 and its inactive analog U73343 (46). Additionally, MTX activated CaNSC are involved in the initiation and progression of Ca2+ overload necrotic/oncotic cell death. The

similarities between MTX-activated CaNSC and TRPM7 raise the intriguing possibility

that TRPM7 may act as an endogenous MTX receptor.

Several studies have shown that Mg2+ inhibits TRPM7 channel activity (124,157).

Therefore, if TRPM7 and the MTX activated channel are the same, it would be expected that MTX-activated channel, like TRPM7, would be sensitive to blockade by Mg2+. To further evaluate the similarity between MTX-activated CaNSC and TRPM7, the Mg2+

2+ sensitivity of both the MTX-induced increases in [Ca ]i and COP activity were

2+ examined. In preliminary experiments, stimulation with 0.05 nM MTX in 5 mM [Ca ]e/

2+ 2+ 1 mM [Mg ]e, leads to an increase in [Ca ]i of ~ 500 nM above baseline within ~ 1 min

2+ (Fig 4.1A). However when [Mg ]e was raised to 20 mM, the MTX-induced rise in

2+ 2+ [Ca ]i under the same conditions only reaches ~100 nM (Fig 4.1A). At 1 mM [Ca ]e,

2+ high [Mg ]e had an even more dramatic effect almost completely blocking the rise in

2+ [Ca ]i. Although other interpretations are possible, this suggests that MTX-activated

CaNSC may be blocked by Mg2+, like TRPM7. Therefore, both TRPM7 and the MTX-

activated channel share the following characteristics; ubiquitous tissue distribution,

blockade by the PLC inhibitors U73122/U73343, and blockade by elevated Mg2+. In

addition both blockade and suppression of TRPM7 expression prevent neuronal cell

death following anoxia, suggesting that TRPM7 is an essential mediator of anoxic cell 137

death (1). Similarly, blockade of the MTX-induced rise in Ca2+ blocks necrotic/oncotic cell death in BAEC cells (46). It should be noted that there are biophysical differences in the TRPM7 and MTX activated channels. Most strikingly, whole cell TRPM7 currents are distinctly outwardly rectifying (1,157), whereas the MTX-activated channels exhibit a linear current voltage relationship (112). However evaluating similarities in channel properties are complicated in that they were recorded under different conditions in different cell types. This fact hinders a comparison between these to channels from the existing literature. A direct electrophysiological comparison of both TRPM7 and MTX- activated channels has not been performed. Therefore it will be necessary to examine the biophysical properties (i.e. rectification, selectivity sequence, permeability ratios, single channel characteristics, etc.) of both whole cell currents and single channels for these channels to determine if the MTX-activated CaNSC and TRPM7 are the same entity.

To further characterize the effect of Mg2+ on the MTX-activated cell death cascade the COP activity was evaluated at both [Mg2+] tested above. Unlike the MTX-

2+ 2+ activated increase in [Ca ]i, COP activity was not affected by increasing [Mg ]e. At 5

2+ mM [Ca ]e, COP activity, as determined by EB uptake, is nearly identical for both 1 and

2+ 20 mM [Mg ]e (3.05 % per min ± 0.871 and 3.30 % per min ± 0.792, respectively) (Fig

4.1B). The delay in the second phase of EB uptake (cell lysis) likely reflects the delayed

2+ 2+ 2+ increase in [Ca ]i in the presence of high Mg . Thus, increasing extracellular Mg

2+ decreases the magnitude of the MTX-induced rise in [Ca ]i 4- to 5-fold while having little effect upon either the kinetics of COP activation or COP activity itself.

The combined results from these preliminary experiments raise the possibility that

2+ MTX activation of TRPM7 causes two independent events. Initially, the rise in [Ca ]i 138

could be a consequence of MTX influencing TRPM7 channel activation or gating. At the

same time, MTX could lead to the induction of the kinase activity of TRPM7 leading to

phosporylation of either COP itself or some intermediate regulatory protein that effects

COP formation/activity. This hypothesis could account for the experimental observation

that blockade of the MTX-activated CaNSC by Mg2+ has no effect upon the initiation of

COP activity. In addition, COP activation is greatly delayed at temperatures below 37 C, suggesting that there may be some enzymatic step, like protein phosphorylation involved in initiation of COP activity (160). To test this hypothesis, it would be valuable to examine the effect of kinase inhibitors or overexpression of protein phsoshatases on COP activation following MTX stimulation. If the kinase activity of TRPM7 does play a role in the initiation of COP formation or COP activity itself, these maneuvers would be expected to decrease COP formation/activity at a given MTX concentration.

The identity of the COP protein is not known at this time. One possible

explanation of COP formation is that the unknown COP protein aggregates to form a larger pores in the plasma membrane. This explanation seems unlikely based upon evidence in the literature. Previous studies using vital dyes of increasing molecular weight have shown that the permeability of these dyes through COP is inversely proportional to dye molecular weight, and that dye uptake is linear during COP (45). This suggests that COP is a pore of fixed dimension of finite permeability. The formation of

COP also occurs following activation of P2X7 purinergic receptors (160). The channels activated by MTX and P2X7 are clearly distinct, however, COP activated by both of these channels is indistinguishable (161). This has led to speculation that COP is a separate membrane associated-pore that is activated by a variety of Ca2+ permeable channels. 139

Interestingly, evidence in the literature has shown that over expression of P2X7 receptors causes a shift in the MTX dose response curve resulting in higher concentrations of MTX necessary to activate a similar level of COP activity (161). This result suggests that

MTX-activated and P2X7-activated channels may be linked to a common pool of COP in

the plasma membrane and that these channels could be coupled to the COP subunits. In

addition, the fact that the MTX-induced cell death cascade occurs in a variety of cell

types and leads to a cell death cascade indistinguishable from that following P2X7 suggests that COP may be part of a common, conserved, biochemical pathway that is initiated following the activation of different CaNSC.

The data presented above suggest that COP: is an independent pore of fixed dimension and finite permeability; can be activated by a variety of different Ca2+ channels; and may be in physical association with these channels. Therefore it will be valuable to determine the identity of the COP protein. One potential approach to identify the COP protein could be to biochemically purify COP via its association with P2X7 or the yet unidentified MTX-activated channels. The observation that overexpression of

P2X7 shifts the MTX dose response curve for COP and therefore it may be in physical

association with COP raises the possibility that P2X7 could be used a tool for the

purification of COP. This would involve co-immunoprecipitation of proteins associated

with P2X7 utilizing an anti-P2X7 antibody. The COP protein would be expected to be present in the lysate and should bind to the P2X7 immunocomplex (based on the assumption that P2X7 or alternatively the MTX channel are physically coupled to COP in

the absence of stimulation). The proteins that remains bound to the P2X7 subunits could the be collected, and characterized both biochemically and functionally. Biochemical 140

assays would included separation of the proteins on the basis of molecular weight using

SDS-PAGE, and then performing mass spectroscopic analysis to obtain sequence

information of the purified proteins. Once candidate COP proteins are identified,

functional assays would include overexpression using the recombinant baculovirus/insect

Sf9 cell expression system. These cells have endogenous MTX activated CaNSC but

have no endogenous COP activity. Overexpression of candidate COP proteins should

allow for the evaluation of COP activity from vital dye uptake studies similar to those

performed in chapter 3.

Determine the identity of the Ca2+ binding proteins that may be involved in the steps of the cell death cascade.

It will be necessary to determine the identity of Ca2+-binding protein(s) involved in

regulating each of the steps in the cell death cascade. There are a multitude of signalling

and regulatory proteins with known Ca2+ binding domains including those of the EF-hand and C2 families (Table 1). Evidence in the literature also indicates that calpains are activated following treatment of neurons with MTX (210) and in capsaicin-induced neurotoxicity (22). It has also been reported that calpain inhibitors can protect against cell death following anoxia and other toxicants both in vivo and in vitro (67,103).

Calpains are a family of ubiquitous Ca2+-dependent proteases that are known to cleave a

variety of substrates involved in cellular adhesion and signalling (50,54,153). In

preliminary studies, I examined the effect of the calpain inhibitor calpeptin on the MTX-

induced cell death cascade. In cells plated onto coverslips, the calpain inhibitor calpeptin

(20 µM) caused a delay in time to cell lysis by ~ 5 min (Fig 4.2). This delay was 141

observed both in loss of cell-associated GFP (Fig 4.2A), and uptake of EB (Fig 4.2B).

Similar results were obtained with calpain inhibitors PD150606 (50 µM) and calpain inhibitor III (10 µM) (data not shown). Further experiments will be necessary to determine the role of calpain in each phase of the cell death cascade. In addition it will be important to determine the identity of other Ca2+ binding proteins involved in the various phases. Identification of these proteins could provide targets for the development of therapeutic modalities that may be useful in the treatment of stroke and myocardial infarction.

142

FIGURES

2+ 2+ Fig 4.1 Effect of Mg on MTX-induced rise in [Ca ]i and EB uptake. Four traces are shown superimposed in each panel. BAECs were suspended in HBS containing either

2+ 2+ 5 (z) or 1 mM [Ca ]e (). In a second trace, [Mg ]e was increased to yield 25 mM

2+ total divalent in either 5 ({) or 1 mM [Ca ]e ( ). MTX (0.05 nM) was added to

BAECs at the time indicated by the arrow and fura-2 fluorescence ratio (Panel A) or

EB fluorescence (Panel B) was measured as a function of time as described

previously in Fig. 3.1. Curves represent mean values of 3 independent experiments;

mean +/- SE values are shown at selected time points.

143

144

Fig 4.2. Effect of calpeptin on loss of cell-associated GFP and EB uptake - single cell

response. The average single cell GFP (Panel A) and EB (Panel B) fluorescence values

were calculated from experiments performed either in the presence ({) or absence (z) of

20 µM calpeptin. In both panels MTX (0.3 nM) was added at t = 5 min. The symbols at selected time points represent mean ± S.E values.

145

146

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