Biomedical subjects

William P Schilling

Publications and source records attributed to William P Schilling.

17 recordsLinked to original sources

PubMed2006

TRPC3 channels colocalize with Na+/Ca2+ exchanger and Na+ pump in axial component of transverse-axial tubular system of rat ventricle.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable, nonselective cation channels activated after stimulation of G protein-coupled membrane receptors linked to phospholipase C (PLC). Although the PLC/inositol phosphate signaling pathway is known to exist in heart, expression and subcellular distribution of TRPC channel proteins in ventricular myocardium have not been evaluated. Of the six members of the TRPC channel family examined here, only TRPC3 was found by Western blot analysis of membrane proteins from rodent or canine ventricle. Likewise, only TRPC3 was observed in immunofluorescence analysis of thin sections from rat ventricle. TRPC3 was also the only family member observed in neonatal rat ventricular myocytes in culture. In longitudinal sections of rat ventricle, TRPC3 was predominantly localized to the intercalated disk region of the myocyte. However, transverse sections through heart muscle or single isolated adult myocytes revealed TRPC3-specific labeling in a vast network of intracellular membranes, where it colocalized with the Na(+)-K(+)-ATPase (NKA) pump and the Na(+)/Ca(2+) exchanger (NCX) but not with the ryanodine receptor or the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Reciprocal immunoprecipitation assays from rat or canine ventricle showed that TRPC3 associates with NKA and NCX but not with the plasmalemmal Ca(2+)-ATPase pump. Immunoprecipitations from Sf9 insect cells heterologously expressing TRPC3, NKA, and NCX in various combinations revealed that NKA and NCX interact and that TRPC3 and NCX interact, but that TRPC3 does not directly associate with NKA. Together, these results suggest that TRPC3 is localized in the ventricular myocyte to the axial component of the transverse-axial tubular system, where it exists in a signaling complex that includes NCX and NKA.

Animals↗

PubMed2006

Palytoxin-induced cell death cascade in bovine aortic endothelial cells.

The plasmalemmal Na(+)-K(+)-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca(2+). However, the ability of PTX to directly increase cytosolic free Ca(2+) concentration ([Ca(2+)](i)) via Na(+) pump channels and to initiate Ca(2+) overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca(2+)](i) and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3-100 nM) produced a graded increase in [Ca(2+)](i) that was dependent on extracellular Ca(2+). The increase in [Ca(2+)](i) initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC(50) < 1 microM. The elevation in [Ca(2+)](i) could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca(2+)](i) occurs via the Na(+) pump. Subsequent to the rise in [Ca(2+)](i), PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca(2+) or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca(2+) overload.

Acrylamides↗

PubMed2006

Human TRPC6 expressed in HEK 293 cells forms non-selective cation channels with limited Ca2+ permeability.

TRPC6 is thought to be a Ca(2+)-permeable cation channel activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C (PLC). TRPC6 current is also activated by exogenous application of 1-oleoyl-acetyl-sn-glycerol (OAG) or by inhibiting 1,2-diacylglycerol (DAG) lipase activity using RHC80267. In the present study, both OAG and RHC80267 increased whole-cell TRPC6 current in cells from a human embryonic kidney cell line (HEK 293) stably expressing TRPC6, but neither compound increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) when the cells were bathed in high-K(+) buffer to hold the membrane potential near 0 mV. These results suggested that TRPC6 channels have limited Ca(2+) permeability relative to monovalent cation permeability and/or that Ca(2+) influx via TRPC6 is greatly attenuated by depolarization. To evaluate Ca(2+) permeability, TRPC6 currents were examined in extracellular buffer in which Ca(2+) was varied from 0.02 to 20 mm. The results were consistent with a pore-permeation model in which Ca(2+) acts primarily as a blocking ion and contributes only a small percentage ( approximately 4%) to whole-cell currents in the presence of extracellular Na(+). Measurement of single-cell fura-2 fluorescence during perforated-patch recording of TRPC6 currents showed that OAG increased [Ca(2+)](i) 50-100 nm when the membrane potential was clamped at between -50 and -80 mV, but had little or no effect if the membrane potential was left uncontrolled. These results suggest that in cells exhibiting a high input resistance, the primary effect of activating TRPC6 will be membrane depolarization. However, in cells able to maintain a hyperpolarized potential (e.g. cells with a large inwardly rectifying or Ca(2+)-activated K(+) current), activation of TRPC6 will lead to a sustained increase in [Ca(2+)](i). Thus, the contribution of TRPC6 current to both the kinetics and magnitude of the Ca(2+) response will be cell specific and dependent upon the complement of other channel types.

Calcium↗

PubMed2005

Identification and localization of TRPC channels in the rat kidney.

It is well established that transient receptor potential (TRP) channels are activated following stimulation of G protein-coupled membrane receptors linked to PLC, but their differential expression in various cells of the renal nephron has not been described. In the present study, immunoprecipitations from rat kidney lysates followed by Western blot analysis using TRPC-specific, affinity-purified antibodies revealed the presence of TRPC1, -C3, and -C6. TRPC4, -C5, and -C7 were nondetectable. TRPC1 immunofluorescence was detected in glomeruli and specific tubular cells of the cortex and outer medulla. TRPC1 colocalized with aquaporin-1, a marker for proximal tubule and thin descending limb, but not with aquaporin-2, a marker for connecting tubule and collecting duct cells. TRPC3 and -C6 immunolabeling was predominantly confined to glomeruli and specific tubular cells of the cortex and both the outer and inner medulla. TRPC3 and -C6 colocalized with aquaporin-2, but not with the Na(+)/Ca(2+) exchanger or peanut lectin. Thus TRPC3 and -C6 proteins are expressed in principle cells of the collecting duct. In polarized cultures of M1 and IMCD-3 collecting duct cells, TRPC3 was localized exclusively to the apical domain, whereas TRPC6 was found in both the basolateral and apical membranes. TRPC3 and TRPC6 were also detected in primary podocyte cultures, whereas TRPC1 was exclusively expressed in mesangial cell cultures. Specific immunopositive labeling for TRPC4, -C5, or -C7 was not observed in kidney sections or cell lines. These results suggest that TRPC1, -C3, and -C6 may play a functional role in PLC-dependent signaling in specific regions of the nephron.

Animals↗

PubMed2005

Proteomic analysis of TRPC5- and TRPC6-binding partners reveals interaction with the plasmalemmal Na(+)/K(+)-ATPase.

Mammalian transient receptor potential canonical (TRPC) genes encode a family of nonselective cation channels that are activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. In Drosophila photoreceptor cells, TRP channels are found in large, multimolecular signaling complexes in association with the PDZ-containing scaffolding protein, INAD. A similar mammalian TRPC "signalplex" has been proposed, but has yet to be defined. In the present study, affinity-purified polyclonal antibodies against TRPC5 and TRPC6 were used to immunoprecipitate signalplex components from rat brain lysates. Immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and sequenced by mass spectrometry. Proteins identified in the immunoprecipitates included cytoskeletal proteins spectrin, myosin, actin, drebrin, tubulin, and neurabin; endocytic vesicle-associated proteins clathrin, dynamin and AP-2; and the plasmalemmal Na(+)/K(+)-ATPase (NKA) pump. Several of these interactions were confirmed by reciprocal immunoprecipitation followed by Western blot analysis. In lysates from rat kidney, TRPC6, but not TRPC3, was found to coimmunoprecipitate with the NKA pump. Likewise, TRPC6, stably expressed in human embryonic kidney (HEK) cells, coimmunoprecipitated with endogenous NKA and colocalized with the pump to the plasmalemma when examined by immunofluorescence microscopy. Cell surface biotinylation experiments in intact HEK cells, confirmed that both the Na(+) pump and TRPC6 were present in the surface membrane and appeared to interact. Lastly, TRPC6 coimmunoprecipitated with the NKA pump when the proteins were coexpressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. These observations suggest that TRPC6 and the Na(+) pump are part of a functional complex that may be involved in ion transport and homeostasis in both the brain and kidney.

Animals↗

PubMed2005

TRPC channels as signal transducers.

The study of the TRPC cation channels as signal transducers in sensory neurons is in its infancy. Mechanoreceptors that monitor arterial pressure are prime candidates for the involvement of TRPC channels as either primary mechanical transducers or as modulators of the transduction process. Their activity patterns can be regulated by growth factors such as BDNF and by a variety of ligands that activate Gq-coupled receptors, mechanisms that have been shown in heterologous expression systems to activate TRPC channels. We investigated the distribution of TRPC1 and TRPC3-7 in nodose sensory neurons and in their peripheral axons that terminate as mechanosensitive receptors in the aortic arch of the rat. Using immunocytochemical techniques we identified these six TRPC proteins in the soma of the nodose neurons but only TRPC1 and TRPC3-5 were found to distribute to the peripheral axons and the mechanosensory terminals. TRPC1 and TRPC3 extended into the low threshold complex sensory endings with very strong labeling. In contrast, TRPC4 and TRPC5 were found primarily in major branches of the receptor but immunoreactivity was weak in the region where mechanotransduction is presumed to occur. Terminals arising from unmyelinated fibers also expressed TRPC1 and TRPC3-5 but not all fibers expressed all of the channels suggesting that specific TRPC protein may be aligned with previously described subclasses of the unmyelinated C-fibers.

Afferent Pathways↗

PubMed2004

Association of immunophilins with mammalian TRPC channels.

Drosophila photoreceptor channels TRP and TRPL are held in a large signalplex by the scaffolding protein, INAD. Immunophilin FKBP59, another member of the signalplex, binds to both INAD and TRPL. Mutation P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709), eliminates TRPL interaction with FKBP59. The first leucylprolyl (LP) dipeptide in this region is conserved in mammalian TRPC channel proteins. However, the second LP is changed to isoleucylprolyl (IP) in TRPC1, -C4, and -C5, and valylprolyl (VP) in TRPC3, -C6, and -C7. The purpose of the present study was to determine if mammalian FKBP12 or FKBP52 interact with TRPC channel proteins. Using TRPC-specific antibodies, immunoprecipitations from Sf9 cells individually co-expressing each of the TRPC proteins along with the immunophilins showed that TRPC3, -C6, and -C7 interact with FKBP12, whereas TRPC1, -C4, and -C5 interact with FKBP52. The binding of FKBP12 and FKBP52 was specific and could be displaced by the immunosuppressant drug FK506, at concentrations of 0.5 and 10 microm, respectively. To evaluate TRPC-immunophilin interactions in vivo, immunoprecipitations were performed using membrane lysates of rat cerebral cortex. FKBP12 co-immunoprecipitated with TRPC3, -C6, and -C7 from rat brain, whereas FKBP52 was found to associate with TRPC1, -C4, and -C5. The association of immunophilins with the TRPC channels in rat brain lysates could be displaced by FK506. Receptor-mediated activation of TRPC6, stably expressed in HEK cells, was significantly inhibited by FK506, which also disrupted interaction between TRPC6 and the endogenous immunophilin found in HEK cells. Pro to Gln mutations in the first LP dipeptide in the putative FKBP binding domain eliminated FKBP12 and FKBP52 interaction with TRPC3 and -C6, and TRPC1 and -C4, respectively. However, mutual swap of VP and IP in TRPC3 and TRPC5 did not alter the association or the selectivity of the channels for their respective immunophilin binding partner. These results suggest that immunophilins are TRPC channel accessory proteins that play an important role in the mechanism of channel activation following receptor stimulation.

Animals↗

PubMed2004

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

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca(2+) overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca(2+)-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules <800 Da to enter the cell. The cells then lyse not by rupture of the plasmalemma but through the activation of a "death" channel that lets large proteins (e.g., 140-160 kDa) leave the cell. These changes in permeability are accompanied by the formation of membrane blebs. In this study, we took advantage of the well-known differences in affinity of various Ca(2+)-binding proteins for Ca(2+) and Sr(2+) vs. Ba(2+) 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 the replacement of Ca(2+) with either Sr(2+) or Ba(2+) delayed both MTX-induced activation of COP, as indicated by the uptake of ethidium bromide, and subsequent cell lysis, as indicated by the uptake of propidium iodide or the release of cell-associated green fluorescent protein. MTX-induced responses were mimicked by ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba(2+) not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca(2+)-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba(2+). Taken together, these results suggest that intracellular high-affinity Ca(2+)-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise.

Animals↗

PubMed2004

Activation of human TRPC6 channels by receptor stimulation.

The human TRPC6 channel was expressed in human embryonic kidney (HEK) cells, and activity was monitored using the giga-seal technique. Whole cell membrane currents with distinctive inward and outward rectification were activated by carbachol (CCh) in TRPC6-expressing cells, but not in lacZ-transfected controls. The effect of CCh was steeply dose-dependent with a K(0.5) of approximately 10 microm and a Hill coefficient of 3-4. A steep concentration-response relationship was also observed when TRPC6 activity was measured using a fluorescence-based imaging plate reader (FLIPR) assay for membrane depolarization. Ionomycin, thapsigargin, and dialysis of the cell with inositol 1,4,5-trisphosphate via the patch pipette had no effect on TRPC6 currents, but exogenous application of 1-oleoyl acetyl-sn-glycerol (OAG, 30-300 microm) produced a slow increase in channel activity. The PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.5 microm) had no significant acute effect on TRPC6, or on the subsequent response to OAG. In contrast, the response to CCh was blocked >90% by PMA pretreatment. To further explore the role of DAG in receptor stimulation, TRPC6 currents were monitored following the sequential addition of CCh and OAG. Surprisingly, concentrations of CCh that produced little or no response in the absence of OAG, produced increases in TRPC6 currents in the presence of OAG that were larger than the sum of either agent alone. Likewise, the response to OAG was superadditive following prior stimulation of the cells with near threshold concentrations of CCh. Overall, these results suggest that generation of DAG alone may not fully account for activation of TRPC6, and that other receptor-mediated events act synergistically with DAG to stimulate channel activity. This synergy may explain, at least in part, the steep dose-response relationship observed for CCh-induced TRPC6 currents expressed in HEK cells.

Calcium Channels↗

PubMed2004

Identification of cytoskeletal [14C]carboplatin-binding proteins reveals reduced expression and disorganization of actin and filamin in cisplatin-resistant cell lines.

Cisplatin resistant (CP-r) cells often show decreased uptake of cisplatin in association with reduced cell surface proteins and decreased endocytosis. In this report, two major [14C]carboplatin-binding proteins were identified as filamin and actin by photoaffinity labeling and mass spectrometry. Decreased expression of these two proteins was found in two different human CP-r cell lines (KB-CP20 and 7404-CP20), in comparison with their parental cell lines (KB-3-1 and BEL-7404), respectively. Disorganization of beta-actin and filamin 250 and 90 was also detected in these CP-r cells by confocal microscopy. Transfection of a wild-type actin-enhanced green fluorescent protein (EGFP) expression vector into 7404-CP20 cells resulted in a nonfilamentous actin-EGFP distribution compared with a normal distribution in the cisplatin-sensitive BEL-7404 cells, suggesting that cytoskeletal organization is disturbed in the CP-r cells. The identification of actin and filamin as [14C]carboplatin-binding proteins and decreased expression and disorganization of several cytoskeletal proteins in CP-r cells provide a molecular and cellular basis for the known defect in endocytosis in these cells.

Actins↗

PubMed2004

Mammalian TRPC channel subunit assembly.

TRPC genes encode a family of ion channel proteins that appear to be responsible for Ca2+ influx following stimulation of membrane receptors linked to phospholipase C. TRPC channels are thought to be tetrameric, and there is growing evidence to suggest heteromultimeric channel assembly. However, the channel subunit composition in vivo and the rules governing subunit assembly remain largely unknown. Like the Drosophila TRP channels, the mammalian TRPCs may reside in large signalling complexes localized to subcellular microdomains by interaction with specific PDZ-containing scaffolding proteins. Selective localization within cellular signalling networks may play an important role in the mode of channel activation following receptor stimulation. Evidence for heteromultimeric TRPC channel assembly gleaned from overexpression studies will be reviewed and recent evidence for the selective association of native TRPC channel subunits in rat brain will be discussed.

Amino Acid Sequence↗

PubMed2004

Distribution of TRPC channels in a visceral sensory pathway.

Until recently most of the published studies addressing the mechanisms of activation of TRPC channels have been carried out in heterologous expression systems. Lack of specific antagonists for the TRPC channels has hampered functional studies of endogenous channels. We approached the role of TRPC channels in native tissue with a study of the distribution of the channel proteins in the carotid chemosensory pathway in the rat. In a previous report we showed that TRPC3/4/5/6 and TRPC7 were present in neurons throughout the petrosal ganglion while TRPC1 was expressed in only a subpopulation of petrosal neurons, at least half of which projected to the carotid body. The TRPC proteins were differentially distributed to the branches of the axons that project centrally to the nucleus of the solitary tract and peripherally to the carotid body. The smallest unmyelinated sensory fibres projecting to the carotid body contained TRPC1/3/4/5 or TRPC6 but not TRPC7. TRPC1 and TRPC3 were concentrated in the larger diameter fibres. Interestingly, only TRPC1 and TRPC4 could be demonstrated in the final terminal endings within glomus cell clusters of the carotid body. In the central axon of the sensory neurons, both TRPC4 and TRPC5 were demonstrated in fibres exiting the solitary tract and projecting to the secondary relay neurons the nucleus of the solitary tract.

Animals↗

PubMed2003

Distribution of transient receptor potential channels in the rat carotid chemosensory pathway.

Glomus cells in the carotid body respond to decreases in oxygen tension of the blood and transmit this sensory information in the carotid sinus nerve to the brain via neurons in the petrosal ganglion. G-protein-coupled membrane receptors linked to phospholipase C may play an important role in this response through the activation of the cation channels formed by the transient receptor potential (TRP) proteins. In the present study, expression of TRPC proteins in the rat carotid body and petrosal ganglion was examined using immunohistochemical techniques. TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 were present in neurons throughout the ganglion. TRPC1 was expressed in only 28% of petrosal neurons, and of this population, 45% were tyrosine hydroxylase (TH)-positive, accounting for essentially all the TH-expressing neurons in the ganglion. Because TH-positive neurons project to the carotid body, this result suggests that TRPC1 is selectively associated with the chemosensory pathway. Confocal images through the carotid body showed that TRPC1/3/4/5/6 proteins localize to the carotid sinus nerve fibers, some of which were immunoreactive to an anti-neurofilament (NF) antibody cocktail. TRPC1 and TRPC3 were present in both NF-positive and NF-negative fibers, whereas TPRC4, TRPC5, and TRPC6 expression was primarily localized to NF-negative fibers. Only TRPC1 and TRPC4 were localized in the afferent nerve terminals that encircle individual glomus cells. TRPC7 was not expressed in sensory fibers. All the TRPC proteins studied were present in type I glomus cells. Although their role as receptor-activated cation channels in the chemosensory pathway is yet to be established, the presence of TRPC channels in glomus cells and sensory nerves of the carotid body suggests a role in facilitating and/or sustaining the hypoxic response.

Animals↗

PubMed2003

Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry.

To evaluate interaction of vanilloid receptor type 1 (TRPV1) with endogenous Ca(2+) signalling mechanisms, TRPV1 was expressed in Spodoptera frugiperda (Sf 9) insect cells using recombinant baculovirus. Stimulation of TRPV1-expressing cells, but not control Sf 9 cells, with resiniferatoxin (RTX), capsaicin or anandamide, produced an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)), with EC(50) values of 166 pM, 24.5 nM and 3.89 microM respectively. In the absence of extracellular Ca(2+), both capsaicin and RTX caused an increase in [Ca(2+)](i) with EC(50) values of approx. 10 microM and 10 nM respectively. This TRPV1-induced release of Ca(2+) 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 Ca(2+) pools, and confocal imaging showed that intracellular TRPV1 immunofluorescence co-localized with the endoplasmic reticulum targeting motif KDEL. To determine if TRPV1-induced mobilization of intracellular Ca(2+) activates endogenous store-operated Ca(2+) entry, the effect of 2-aminoethoxydiphenyl borate (2-APB) on Ba(2+) influx was examined. 2-APB blocked thapsigargin-induced Ba(2+) influx, but not RTX-induced Ba(2+) entry. In the combined presence of thapsigargin and a store-releasing concentration of RTX, the 2-APB-sensitive component was essentially identical with the thapsigargin-induced component. Similar results were obtained in HEK-293 cells stably expressing TRPV1. These results suggest that TRPV1 forms agonist-sensitive channels in the endoplasmic reticulum, which when activated, release Ca(2+) from internal stores, but fail to activate endogenous store-operated Ca(2+) entry. Selective activation of intracellular TRPV1, without concomitant involvement of plasmalemmal Ca(2+) influx mechanisms, could play an important role in Ca(2+) signalling within specific subcellular microdomains.

Animals↗

PubMed2002

Blockade of maitotoxin-induced endothelial cell lysis by glycine and L-alanine.

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs) is a model for oncotic/necrotic cell death. The cascade is initiated by an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which is followed by the biphasic uptake of vital dyes. The initial phase of dye entry reflects activation of large pores and correlates with surface membrane bleb formation; the second phase reflects cell lysis. In the present study, the effect of the cytoprotective amino acid glycine was examined. Glycine had no effect on MTX-induced change in [Ca(2+)](i) or on the first phase of vital dye uptake but produced a concentration-dependent (EC(50) approximately 1 mM) inhibition of the second phase of dye uptake. No cytoprotective effect was observed with l-valine, l-proline, or d-alanine, whereas l-alanine was equieffective to glycine. Furthermore, glycine had no effect on MTX-induced bleb formation. To test the hypothesis that glycine specifically blocks formation of a lytic "pore," the loss of fluorescence from BAECs transiently expressing GFP and concatemers of GFP ranging in size from 27 to 162 kDa was examined using time-lapse videomicroscopy. MTX-induced loss of GFP was rapid, correlated with the second phase of dye uptake, and was relatively independent of molecular size. The MTX-induced loss of GFP from BAECs was completely blocked by glycine. The data suggest that the second "lytic" phase of MTX-induced endothelial cell death reflects formation of a novel permeability pathway that allows macromolecules such as GFP or LDH to escape, yet can be prevented by the cytoprotective agents glycine and l-alanine.

Alanine↗

PubMed2002

Selective association of TRPC channel subunits in rat brain synaptosomes.

TRPC genes encode a ubiquitous family of ion channel proteins responsible for Ca(2+) influx following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. These channels may be localized to large multimeric signaling complexes via association with PDZ-containing scaffolding proteins. Based on sequence homology, the TRPC channel family can be divided into two major subgroups: TRPC1, -C4, and -C5 and TRPC3, -C6, and -C7. Although TRPC channels are thought to be tetramers, the actual subunit composition remains unknown. To determine subunit arrangement, individual TRPC channel pairs were heterologously expressed in Sf9 insect cells and immunoprecipitated using affinity-purified rabbit polyclonal antibodies specific for each channel subtype. Reciprocal co-immunoprecipitations showed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate but that cross-association between the two major subgroups does not occur. Additionally, the interaction between each TRPC channel and the PDZ-containing protein, INAD (protein responsible for the inactivation-no-after-potential Drosophila mutant), was examined. TRPC1, -C4, and -C5 co-immunoprecipitated with INAD, whereas TRPC3, -C6, and -C7 did not. To define channel subunit interactions in vivo, immunoprecipitations were performed from isolated rat brain synaptosomal preparations. The results revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur. These results demonstrate that TRPC channels are present in nerve terminals and provide the first direct evidence for selective assembly of channel subunits in vivo.

Animals↗

PubMed2002

Blockade of maitotoxin-induced oncotic cell death reveals zeiosis.

BACKGROUND: Maitotoxin (MTX) initiates cell death by sequentially activating 1) Ca2+ influx via non-selective cation channels, 2) uptake of vital dyes via formation of large pores, and 3) release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC) in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells. RESULTS: Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC50 asymptotically equal to 1.9 and 0.66 microM, respectively) suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program. CONCLUSIONS: Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms.

Animals↗