TRPV4 mediates tumor-derived endothelial cell migration via arachidonic acid-activated actin remodeling.

Changes in intracellular calcium [Ca(2+)](i) levels control critical cytosolic and nuclear events that are involved in the initiation and progression of tumor angiogenesis in endothelial cells (ECs). Therefore, the mechanism(s) involved in agonist-induced Ca(2+)(i) signaling is a potentially important molecular target for controlling angiogenesis and tumor growth. Several studies have shown that blood vessels in tumors differ from normal vessels in their morphology, blood flow and permeability. We had previously reported a key role for arachidonic acid (AA)-mediated Ca(2+) entry in the initial stages of tumor angiogenesis in vitro. In this study we assessed the mechanism involved in AA-induced EC migration. We report that TRPV4, an AA-activated channel, is differentially expressed in EC derived from human breast carcinomas (BTEC) as compared with 'normal' EC (HMVEC). BTEC display a significant increase in TRPV4 expression, which was correlated with greater Ca(2+) entry, induced by AA or 4αPDD (a selective TRPV4 agonist) in the tumor-derived ECs. Wound-healing assays revealed a key role of TRPV4 in regulating cell migration of BTEC but not HMVEC. Knockdown of TRPV4 expression completely abolished AA-induced BTEC migration, suggesting that TRPV4 mediates the pro-angiogenic effects promoted by AA. Furthermore, pre-incubation of BTEC with AA induced actin remodeling and a subsequent increase in the surface expression of TRPV4. This was consistent with the increased plasma membrane localization of TRPV4 and higher AA-stimulated Ca(2+) entry in the migrating cells. Together, the data presented herein demonstrate that: (1) TRPV4 is differentially expressed in tumor-derived versus 'normal' EC; (2) TRPV4 has a critical role in the migration of tumor-derived but not 'normal' EC migration; and (3) AA induces actin remodeling in BTEC, resulting in a corresponding increase of TRPV4 expression in the plasma membrane. We suggest that the latter is critical for migration of EC and thus in promoting angiogenesis and tumor growth.

Stabilization of cortical actin induces internalization of transient receptor potential 3 (Trp3)-associated caveolar Ca2+ signaling complex and loss of Ca2+ influx without disruption of Trp3-inositol trisphosphate receptor association.

Ca(2+) influx via plasma membrane Trp3 channels is proposed to be regulated by a reversible interaction with inositol trisphosphate receptor (IP(3)R) in the endoplasmic reticulum. Condensation of the cortical actin layer has been suggested to physically disrupt this interaction and inhibit Trp3-mediated Ca(2+) influx. This study examines the effect of cytoskeletal reorganization on the localization and function of Trp3 and key Ca(2+) signaling proteins. Calyculin-A treatment resulted in formation of condensed actin layer at the plasma membrane; internalization of Trp3, Galpha(q/11), phospholipase Cbeta, and caveolin-1; and attenuation of 1-oleoyl-2-acetyl-sn-glycerol- and ATP-stimulated Sr(2+) influx. Importantly, Trp3 and IP(3)R-3 remained co-localized inside the cell and were co-immunoprecipitated. Jasplakinolide also induced internalization of Trp3 and caveolin-1. Pretreatment of cells with cytochalasin D or staurosporine did not affect Trp3 but prevented calyculin-A-induced effects. Based on these data, we suggest that Trp3 is assembled in a caveolar Ca(2+) signaling complex with IP(3)R, SERCA, Galpha(q/11), phospholipase Cbeta, caveolin-1, and ezrin. Furthermore, our data demonstrate that conditions which stabilize cortical actin induce loss of Trp3 activity due to internalization of the Trp3-signaling complex, not disruption of IP(3)R-Trp3 interaction. This suggests that localization of the Trp3-associated signaling complex, rather than Trp3-IP(3)R coupling, depends on the status of the actin cytoskeleton.

Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains.

Trp1 has been proposed as a component of the store-operated Ca(2+) entry (SOC) channel. However, neither the molecular mechanism of SOC nor the role of Trp in this process is yet understood. We have examined possible molecular interactions involved in the regulation of SOC and Trp1 and report here for the first time that Trp1 is assembled in signaling complex associated with caveolin-scaffolding lipid raft domains. Endogenous hTrp1 and caveolin-1 were present in low density fractions of Triton X-100-extracted human submandibular gland cell membranes. Depletion of plasma membrane cholesterol increased Triton X-100 solubility of Trp1 and inhibited carbachol-stimulated Ca(2+) signaling. Importantly, thapsigargin stimulated Ca(2+) influx, but not internal Ca(2+) release, and inositol 1,4,5-triphosphate (IP(3))-stimulated I(soc) were also attenuated. Furthermore, both anti-Trp1 and anti-caveolin-1 antibodies co-immunoprecipitated hTrp1, caveolin-1, Galpha(q/11), and IP(3) receptor-type 3 (IP(3)R3). These results demonstrate that caveolar microdomains provide a scaffold for (i) assembly of key Ca(2+) signaling proteins into a complex and (ii) coordination of the molecular interactions leading to the activation of SOC. Importantly, we have shown that Trp1 is also localized in this microdomain where it interacts with one or more components of this complex, including IP(3)R3. This finding is potentially important in elucidating the physiological function of Trp.

Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells.

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Characteristics of a low affinity passive Ca2+ influx component in rat parotid gland basolateral plasma membrane vesicles.

We have previously reported the presence of two Ca2+ influx components with relatively high (KCa = 152 +/- 79 microM) and low (KCa = 2.4 +/- 0.9 mM) affinities for Ca2+ in internal Ca2+ pool-depleted rat parotid acinar cells [Chauthaiwale et al. (1996) Pfluegers Arch. 432: 105-111]. We have also reported the presence of a high affinity Ca2+ influx component with KCa = 279 +/- 43 microM in rat parotid gland basolateral plasma membrane vesicles (BLMV). [Lockwich, Kim & Ambudkar (1994) J. Membrane Biol. 141:289-296]. The present studies show that a low affinity Ca2+ influx component is also present in BLMV with KCa = 2.3 +/- 0.41 mM (Vmax = 16.36 +/- 4.11 nmoles of Ca2+/mg protein/min). Our data demonstrate that this low affinity component is similar to the low affinity Ca2+ influx component that is activated by internal Ca2+ store depletion in dispersed parotid gland acini by the following criteria: (i) similar KCa for calcium flux, (ii) similar IC50 for inhibition by Ni2+ and Zn2+; (iii) increase in KCa at high external K+, (iv) similar effects of external pH. The high affinity Ca2+ influx in cells is different from the low affinity Ca2+ influx component cells in its sensitivity to pH, KCl, Zn2+ and Ni2+. The low and high affinity Ca2+ influx components in BLMV can also be distinguished from each other based on the effects of Zn2+, Ni2+, KCl, and dicyclohexylcarbodiimide. In aggregate, these data demonstrate the presence of a low affinity passive Ca2+ influx pathway in BLMV which displays characteristics similar to the low affinity Ca2+ influx component detected in parotid acinar cells following internal Ca2+ store depletion.

Reconstitution of a passive Ca(2+)-transport pathway from the basolateral plasma membrane of rat parotid gland acinar cells.

We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca2+ transport pathway and an unsaturable Ca2+ flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289-296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3-5-fold higher 45Ca2+ influx than control liposomes (without protein). Ca2+ uptake into PrL was dependent on the [protein] in PrL and steady state [Ca2+] in PrL was in equilibrium with external [Ca2+]. These data demonstrate that a passive, protein-mediated Ca2+ transport has been reconstituted from BLMV into PrL. 45Ca2+ influx into liposomes did not saturate with increasing [Ca2+] in the assay medium. In contrast, PrL displayed saturable 45Ca2+ influx and exhibited a single Ca2+ flux component with an apparent KCa = 242 +/- 50.9 microM and Vmax = 13.5 +/- 1.14 nmoles Ca2+/mg protein/ minute. The KCa of Ca(2+)-transport in PrL was similar to that of the high affinity Ca2+ influx component in BLMV while the Vmax was about 4-fold higher. The unsaturable Ca2+ flux component was not detected in PrL. 45Ca2+ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn2+ > Mn2+ > Co2+ = Ni2+, and appeared to be more sensitive to lower concentrations of Zn2+ than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca2+ transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40-50% decrease in Vmax of Ca2+ transport without an alteration in KCa. These data strongly suggest that the high affinity, passive Ca2+ transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca2+ influx in the rat parotid gland basolateral plasma membrane.

Temperature-dependent modification of divalent cation flux in the rat parotid gland basolateral membrane.

Divalent cation (Mn2+, Ca2+) entry into rat parotid acinar cells is stimulated by the release of Ca2+ from the internal agonist-sensitive Ca2+ pool via a mechanism which is not yet defined. This study examines the effect of temperature on Mn2+ influx into internal Ca2+ pool-depleted acini (depl-acini, as a result of carbachol stimulation of acini in a Ca(2+)-free medium for 10 min) and passive 45Ca2+ influx in basolateral membrane vesicles (BLMV). Mn2+ entry into depl-acini was decreased when the incubation temperature was lowered from 37 to 4 degrees C. At 4 degrees C, Mn2+ entry appeared to be inactivated since it was not increased by raising extracellular [Mn2+] from 50 microM up to 1 mM. The Arrhenius plot of depletion-activated Mn2+ entry between 37 and 8 degrees C was nonlinear, with a change in the slope at about 21 degrees C. The activation energy (Ea) increased from 10 kcal/mol (Q10 = 1.7) at 21-37 degrees C to 25 kcal/mol (Q10 = 3.0) at 21-8 degrees C. Under the same conditions, Mn2+ entry into basal (unstimulated) cells and ionomycin (5 microM) permeabilized depl-acini exhibit a linear decrease, with Ea of 7.8 kcal/mol (Q10 = 1.5) and 6.2 kcal/mol (Q10 < 1.5), respectively. These data suggest that depletion-activated Mn2+ entry into parotid acini is regulated by a mechanism which is strongly temperature dependent and distinct from Mn2+ entry into unstimulated acini.(ABSTRACT TRUNCATED AT 250 WORDS)

Uncoupling of occlusion from ATP hydrolysis activity in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu3+ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Ca2+ and at 25 degrees C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca2+, 3 mM ATP, and at 25 degrees C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu3+. Digest past TD2 in the presence of 5 mM Ca2+ and 3 mM AMP-PNP (a non-hydrolyzable ATP analog) at 25 degrees C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca2+ transport from ATPase activity is possible by tryptic digestion of the (Ca2+ + Mg2+)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.

Involvement of carboxyl groups in the divalent cation permeability of rat parotid gland basolateral plasma membrane.

Divalent cation permeability of rat parotid gland basolateral plasma membranes was examined in dispersed parotid acini (by Ca2+ or Mn2+ entry) and in isolated basolateral plasma membrane vesicles (BLMV, by 45Ca2+ influx). Mn2+ entry (fura2 quenching) was about 1.6 fold higher in internal Ca2+ pool-depleted acini (Ca(2+)-depl acini) than in unstimulated cells. Mn2+ entry into Ca(2+)-depl acini was increased at external pH > 7.4 and decreased at pH < 7.4. Pretreatment of Ca(2+)-depl acini with the relatively hydrophobic carboxylic group reagent, N,N'-dicyclohexylcarbodiimide (DCCD, 50 microM for 30 min) resulted in the inhibition of Mn2+ entry into Ca(2+)-depl acini to unstimulated levels. Another hydrophobic carboxyl group reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and the relatively hydrophilic carboxyl group reagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMCD) did not affect Mn2+ entry. Similar to the effects in intact acini, Ca2+ influx into BLMV was decreased when the external pH was lowered below 7.4. Also DCCD (5 mM, 30 min), but not EEDQ, decreased (40%) Ca2+ influx in BLMV. However, unlike in acini, the hydrophilic reagents, EDC, EAC, and CMCD decreased Ca2+ permeability in BLMV and the effects were nonadditive with the decrease induced by DCCD. The aggregate effects of carboxyl group reagents on the Ca2+ and Mn2+ permeability in BLMV and intact acini, respectively, suggest that a critical carboxyl group (most likely accessible from the cytoplasmic side of the plasma membrane) is involved in divalent cation flux in rat parotid acinar cells.

Ca2+ permeability of rat parotid gland basolateral plasma membrane vesicles is modulated by membrane potential and extravesicular [Ca2+].

This study examines the Ca2+ permeability of basolateral plasma membrane vesicles (BLMVs) isolated from the rat parotid gland by monitoring the rate of 45Ca2+ efflux from actively-loaded (via the Ca(2+)-ATPase) inside-out BLMVs. Ca2+ efflux from BLMVs into a K(+)-gluconate medium which hyperpolarizes the cytoplasmic side (i.e. outside) of the inside-out BLMVs resulted in a faster rate of Ca2+ efflux compared with a control medium containing N-methyl-D-glucamine (NMDG)-gluconate. Conversely, Ca2+ efflux into a medium which depolarizes the cytoplasmic side of the BLMVs (NMDG-chloride) resulted in slower rates of efflux compared with those observed with the control medium. This increased rate of 45Ca2+ efflux from the hyperpolarized BLMV was inhibited by 1 mM Ni2+, yielding a rate of efflux similar to the rate observed in depolarized BLMVs. The rate of Ca2+ efflux from BLMVs was affected by [Ca2+]o ([Ca2+] on the extravesicular, cytoplasmic side of the vesicle). When [Ca2+]o was kept > 200 nM during efflux, the rate of Ca2+ efflux from both hyper- and depolarized BLMVs was slow and relatively unresponsive to changes in [Ca2+]o, despite sizeable changes in the Ca2+ gradient across the BLMV. However, when [Ca2+]o was lowered < 200 nM, there was an abrupt increase in the rate of Ca2+ efflux from both hyper- and depolarized BLMVs. Additionally, when [Ca2+] was < 200 nM, the rate of Ca2+ efflux appeared to be more sensitive to driving force changes. These data suggest that Ca2+ permeability across the rat parotid gland basolateral plasma membrane is modulated by membrane potential and [Ca2+] on the cytoplasmic side.

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

In reconstituted rabbit skeletal muscle (Ca2+ + Mg2+)-ATPase proteoliposomes, Ca(2+)-uptake is decreased by more than 90% with T2 cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T3 cleavage, which represents the cleavage of A1 fragment to A1a + A1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca2+ efflux between control SR and tryptically digested SR in the absence of Mg2+ + ruthenium red or in the presence of ATP. However, the passive Ca2+ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg2+ + ruthenium red. These results show that the Ca2+ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca2+ channel inhibitors, Mg2+ and ruthenium red. In the reconstituted proteoliposomes, the Ca2+ efflux rates are the same regardless of digestion (T2); also, efflux is not affected by the presence or absence of Mg2+ + ruthenium red. These results indicate that T2 cleavage causes 'uncoupling' of the 'Ca(2+)-pump' from ATP hydrolytic activity. A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca2+ + Mg2+)-ATPase polypeptide with the loss of Ca(2+)-transport. Fits of the theoretical equations to the data are consistent with that Ca(2+)-transport system appears to require a dimer of the polypeptide (Ca2+ + Mg2+)-ATPase.

Tertiary structure and energy coupling in Ca2(+)-pump system.

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H2O molecules surrounding the Ca2+ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca2+ - Ca2+ intersite distance is estimated to be 8-9 A and the average distance from the Ca2+ sites to CrATP is about 18 A. Digestion of the (Ca2+ + Mg2+)-ATPase at the T2 site (Arg 198) causes uncoupling of Ca2(+)-transport from ATPase activity while calcium occlusion due to E1-P formation remains unchanged. Further tryptic digestion beyond T2 and in the presence of ATP diminishes Ca2+ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T2 and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca2+ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.

Identification of a spectroscopic marker for the Ca2(+)-binding site of (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum in the occluded state.

The 7F0----5D0 excitation spectrum of Eu3+ bound to the high-affinity calcium sites of SR (Ca2+ + Mg2+)-ATPase diminishes upon occlusion of the Eu3+ into the interior of the enzyme. This "quenching" was found to be caused by the enzyme itself because trypsin digestion could relieve it. The level of digestion needed to relieve the quenching is beyond the level needed to eliminate occlusion; thus, the two processes are not related. Ca2+ is required during digestion to preserve the quenching, indicating close proximity between the Ca2+ site(s) and the quenching segment. Synthetic peptides were found that could mimic the native enzyme's ability to quench the Eu3+ fluorescence, although no native sequence has yet been identified that could emulate the enzyme.

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

The two high affinity calcium binding sites of the cardiac (Ca2+ + Mg2+)-ATPase have been identified with the use of Eu3+. Eu3+ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H2O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca2+ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6-8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.