Lipid phosphate phosphatase-1 expression in cancer cells attenuates tumor growth and metastasis in mice

Lipid phosphate phosphatase-1 (LPP1) degrades lysophosphatidate (LPA) and attenuates receptor-mediated signaling. LPP1 expression is low in many cancer cells and tumors compared with normal tissues. It was hypothesized from studies with cultured cells that increasing LPP1 activity would decrease tumor growth and metastasis. This hypothesis has never been tested in vivo. To do this, we inducibly expressed LPP1 or a catalytically inactive mutant in cancer cells. Expressing active LPP1 increased extracellular LPA degradation by 5-fold. It also decreased the stimulation of Ca²⁺ transients by LPA, a nondephosphorylatable LPA_1/2 receptor agonist and a protease-activated receptor-1 peptide. The latter results demonstrate that LPP1 has effects downstream of receptor activation. Decreased Ca²⁺ mobilization and Rho activation contributed to the effects of LPP1 in attenuating the LPA-induced migration of MDA-MB-231 breast cancer cells and their growth in 3D culture. Increasing LPP1 expression in breast and thyroid cancer cells decreased tumor growth and the metastasis by up to 80% compared with expression of inactive LPP1 or green fluorescent protein in syngeneic and xenograft mouse models. The present work demonstrates for the first time that increasing the LPP1 activity in three lines of aggressive cancer cells decreases their abilities to produce tumors and metastases in mice.     

Enzymological properties of the LPP1-encoded lipid phosphatase from Saccharomyces cerevisiae

The product of the LPP1 gene in Saccharomyces cerevisiae is a membrane-associated enzyme that catalyzes the Mg(2+)-independent dephosphorylation of phosphatidate (PA), diacylglycerol pyrophosphate (DGPP), and lysophosphatidate (LPA). The LPP1-encoded lipid phosphatase was overexpressed 681-fold in Sf-9 insect cells and used to examine the enzymological properties of the enzyme using PA, DGPP, and LPA as substrates. The optimum pH values for PA phosphatase, DGPP phosphatase, and LPA phosphatase activities were 7. 5, 7.0, and 7.0, respectively. Divalent cations (Mn(2+), Co(2+), and Ca(2+)), NaF, heavy metals, propranolol, phenylglyoxal, and N-ethylmaleimide inhibited the PA phosphatase, DGPP phosphatase, and LPA phosphatase activities of the enzyme. The inhibitory effects of N-ethylmaleimide and phenylglyoxal on the LPP1-encoded enzyme were novel properties when compared with other Mg(2+)-independent lipid phosphate phosphatases from S. cerevisiae and mammalian cells. The LPP1-encoded enzyme exhibited saturation kinetics with respect to the surface concentrations of PA (K(m)=0.05 mol%), DGPP (K(m)=0.07 mol%), and LPA (K(m)=0.08 mol%). Based on specificity constants (V(max)/K(m)LPA (1.3 units/mg/mol%). DGPP (K(i)=0.12 mol%) was a competitive inhibitor with respect to PA, and PA (K(i)=0.12 mol%) was a competitive inhibitor with respect to DGPP. This suggested that the binding sites for these substrates were the same. The enzymological properties of the LPP1-encoded enzyme differed significantly from those of the S. cerevisiae DPP1-encoded lipid phosphatase, a related enzyme that also utilizes PA, DGPP, and LPA as substrates.     

Lipid phosphate phosphatase-1 and Ca2+ control lysophosphatidate signaling through EDG-2 receptors

The serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through the EDG family of G protein-coupled receptors. The present study investigated mechanisms by which dephosphorylation of exogenous LPA by lipid phosphate phosphatase-1 (LPP-1) controls cell signaling. Overexpressing LPP-1 decreased the net specific cell association of LPA with Rat2 fibroblasts by approximately 50% at 37 degrees C when less than 10% of LPA was dephosphorylated. This attenuated cell activation as indicated by diminished responses, including cAMP, Ca(2+), activation of phospholipase D and ERK, DNA synthesis, and cell division. Conversely, decreasing LPP-1 expression increased net LPA association, ERK stimulation, and DNA synthesis. Whereas changing LPP-1 expression did not alter the apparent K(d) and B(max) for LPA binding at 4 degrees C, increasing Ca(2+) from 0 to 50 micrometer increased the K(d) from 40 to 900 nm. Decreasing extracellular Ca(2+) from 1.8 mm to 10 micrometer increased LPA binding by 20-fold, shifting the threshold for ERK activation to the nanomolar range. Hence the Ca(2+) dependence of the apparent K(d) values explains the long-standing discrepancy of why micromolar LPA is often needed to activate cells at physiological Ca(2+) levels. In addition, the work demonstrates that LPP-1 can regulate specific LPA association with cells without significantly depleting bulk LPA concentrations in the extracellular medium. This identifies a novel mechanism for controlling EDG-2 receptor activation.     

Lysophospholipid receptor-dependent and -independent calcium signaling

Changes in cellular Ca(2+) concentrations form a ubiquitous signal regulating numerous processes such as fertilization, differentiation, proliferation, contraction, and secretion. The Ca(2+) signal, highly organized in space and time, is generated by the cellular Ca(2+) signaling toolkit. Lysophospholipids, such as sphingosine-1-phosphate (S1P), sphingosylphosphorylcholine (SPC), or lysophosphatidic acid (LPA) use this toolkit in a specific manner to initiate their cellular responses. Acting as agonists at G protein-coupled receptors, S1P, SPC, and LPA increase the intracellular free Ca(2+) concentration ([Ca(2+)](i)) by using the classical, phospholipase C (PLC)-dependent pathway as well as PLC-independent pathways such as sphingosine kinase (SphK)/S1P. The S1P(1) receptor, via protein kinase C, inhibits the [Ca(2+)](i) transients caused by other receptors. Both S1P and SPC also act intracellularly to regulate [Ca(2+)](i). Intracellular S1P mobilizes Ca(2+) in intact cells independently of G protein-coupled S1P receptors, and Ca(2+) signaling by many agonists requires SphK-mediated S1P production. As shown for the FcepsilonRI receptor, PLC and SphK may contribute specific components to the overall [Ca(2+)](i) transient. Of the many open questions, identification of the intracellular S1P target site(s) appears to be of particular importance.     

Lysophosphatidic acid is a mediator of Trp-Lys-Tyr-Met-Val-d-Met-induced calcium influx

Intracellular calcium (Ca(2+)) homeostasis is very strictly regulated, and the activation of G-protein-coupled receptor (GPCR) can cause two different calcium changes, intracellular calcium release, and calcium influx. In this study, we investigated the possible role of lysophosphatidic acid (LPA) on GPCR-induced Ca(2+) signaling. The addition of exogenous LPA induced dramatic Ca(2+) influx but not intracellular Ca(2+) release in U937 cells. LPA-induced Ca(2+) influx was not affected by pertussis toxin and phospholipase C inhibitor (U73122), ruling out the involvement of pertussis toxin-sensitive G-proteins, and phospholipase C. Stimulation of U937 cells with Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), which binds to formyl peptide receptor like 1, enhanced phospholipase A(2) and phospholipase D activation, indicating LPA formation. The inhibition of LPA synthesis by phospholipase A(2)-specific inhibitor (MAFP) or n-butanol significantly inhibited WKYMVm-induced Ca(2+) influx, suggesting a crucial role for LPA in the process. Taken together, we suggest that LPA mediates WKYMVm-induced Ca(2+) influx.     

Subtype-specific residues involved in ligand activation of the endothelial differentiation gene family lysophosphatidic acid receptors

Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA(1-3). We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA(1-3) predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E(max)) and potency (EC(50)) of LPA-elicited Ca(2+) transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA(1-3) and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA(1) and LPA(2) more than in LPA(3). The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA(1) and LPA(2) but reduced the activation and modeled interactions of LPA(3). The R5.38A mutant of LPA(2) and R5.38N mutant of LPA(3) showed diminished activation by LPA; however, in LPA(1) the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA(2), K7.36A decreased the potency of LPA; in LPA(1) this same mutation increased the E(max). In LPA(3), R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC(50) in response to LPA 10-fold. In LPA(1-3), the mutation Q3.29E caused a modest increase in EC(50) in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA(2) and LPA(3) receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors.     

Gintonin, newly identified compounds from ginseng, is novel lysophosphatidic acids-protein complexes and activates G protein-coupled lysophosphatidic acid receptors with high affinity

Recently, we isolated a subset of glycolipoproteins from Panax ginseng, that we designated gintonin, and demonstrated that it induced [Ca2+]i transients in cells via G protein-coupled receptor (GPCR) signaling pathway(s). However, active components responsible for Ca2+ mobilization and the corresponding receptor(s) were unknown. Active component(s) for [Ca2+]i transients of gintonin were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry and ion-mobility mass spectrometry, respectively. The corresponding receptor(s)were investigated through gene expression assays. We found that gintonin contains LPA C18:2 and other LPAs. Proteomic analysis showed that ginseng major latex-like protein and ribonuclease-like storage proteins are protein components of gintonin. Gintonin induced [Ca2+]i transients in B103 rat neuroblastoma cells transfected with human LPA receptors with high affinity in order of LPA2 >LPA5 > LPA1 > LPA3 > LPA4. The LPA1/LPA3 receptor antagonist Ki16425 blocked gintonin action in cells expressing LPA1 or LPA3. Mutations of binding sites in the LPA3 receptor attenuated gintonin action. Gintonin acted via pertussis toxin (PTX)-sensitive and -insensitive G protein-phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3)-Ca2+ pathways. However, gintonin had no effects on other receptors examined. In human umbilical vein endothelial cells (HUVECs) gintonin stimulated cell proliferation and migration. Gintonin stimulated ERK1/2 phosphorylation. PTX blocked gintonin-mediated migration and ERK1/2 phosphorylation. In PC12 cells gintonin induced morphological changes, which were blocked by Rho kinase inhibitorY-27632. Gintonin contains GPCR ligand LPAs in complexes with ginseng proteins and could be useful in the development of drugs targeting LPA receptors.     

Dioleoyl phosphatidic acid increases intracellular Ca2+ through endogenous LPA receptors in C6 glioma and L2071 fibroblasts

Phosphatidic acid (PA) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in C6 rat glioma and L2071 mouse fibroblast cells. Dioleoyl PA (PA, 18:1) was the most efficacious, followed by dipalmitoyl PA (16:0 PA) and dimyristoyl PA (14:0 PA). Lysophosphatidic acid (LPA) also increased the [Ca(2+)](i) in the both cells. PA desensitized LPA-induced Ca(2+) response completely in C6 cells, but partly in L2071 cells. Treatment of pertussis toxin (PTX), a specific inhibitor of G(i/o)-type G proteins, completely ameliorated LPA- and PA-induced Ca(2+) response in C6 cells. However, in L2071 cells, PTX inhibited PA-induced Ca(2+) increase by 80% and LPA-induced one by 20%. Ki16425, a specific inhibitor of LPA(1)/LPA(3) receptors, completely inhibited both LPA- and PA-induced Ca(2+) responses in C6 cells. On the other hand, in L2071 cells, Ki16425 completely inhibited PA-induced Ca(2+) response, but partly LPA-induced one. VPC32183, another specific inhibitor of LPA(1)/LPA(3) receptors, completely inhibited LPA- and PA-induced Ca(2+) responses in both C6 and L2071 cells. Therefore, PA and LPA appear to increase [Ca(2+)](i) through Ki16425/VPC32183-sensitive LPA receptor coupled to PTX-sensitive G proteins in C6 cells. In L2071 cells, however, LPA increases [Ca(2+)](i) through Ki16425-insensitive LPA receptor coupled to PTX-insensitive G proteins and Ki16425-sensitive LPA receptor coupled to PTX-sensitive G protein, whereas PA utilized only the latter pathway. Our results suggest that PA acts as a partial agonist on endogenous LPA receptors, which are sensitive to Ki16425 and coupled to PTX-sensitive G protein, but not on LPA receptors, which are not sensitive to Ki16425 and coupled to PTX-insensitive G protein.     

Mechanisms of the lysophosphatidic acid-induced increase in [Ca(2+)](i) in skeletal muscle cells.

Although lysophosphatidic acid (LPA) is known to increase intracellularfree calcium concentration ([Ca(2+)](i)) in different cell types, the effect of LPA on the skeletal muscle cells is not known. The present study was therefore undertaken to examine the effect of LPA on the [Ca(2+)](i) in C2C12 cells. LPA induced a concentration and time dependent increase in [Ca(2+)](i), which was inhibited by VPC12249, VPC 32183 and dioctanoyl glycerol pyrophosphate, LPA1/3 receptor antagonists. Pertussis toxin, a G(i) protein inhibitor, also inhibited the LPA-induced increase in [Ca(2+)](i). Inhibition of tyrosine kinase activities with tyrphostin A9 and genistein also prevented the increase in [Ca(2+)](i) due to LPA. Likewise, wortmannin and LY 294002, phosphatidylinositol 3-kinase (PI3-K) inhibitors, inhibited [Ca(2+)](i) response to LPA. The LPA effect was also attenuated by ethylene glycolbis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), an extracellular Ca(2+) chelator, Ni(2+) and KB-R7943, inhibitors of the Na(+)-Ca(2+) exchanger; the receptor operated Ca(2+) channel (ROC) blockers, 2-aminoethoxydiphenyl borate and SK&F 96365. However, the L-type Ca(2+) channel blockers, verapamil and diltiazem; the store operated Ca(2+) channel blockers, La(3+) and Gd(3+); a sarcoplasmic reticulum calcium pump inhibitor, thapsigargin; an inositol trisphosphate receptor antagonist, xestospongin and a phospholipase C inhibitor, U73122, did not prevent the increase [Ca(2+)](i) due to LPA. Our data suggest that the LPA-induced increase in [Ca(2+)](i) might occur through G(i)-protein coupled LPA(1/3) receptors that may be linked to tyrosine kinase and PI3-K, and may also involve the Na(+)-Ca(2+) exchanger as well as the ROC. In addition, LPA stimulated C2C12 cell proliferation via PI3-K. Thus, LPA may be an important phospholipid in the regulation of [Ca(2+)](i) and growth of skeletal muscle cells.     

Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid.

Lysophosphatidic acid (LPA), together with sphingosine 1-phosphate, is a bioactive lipid mediator that acts on G-protein-coupled receptors to evoke multiple cellular responses, including Ca(2+) mobilization, modulation of adenylyl cyclase, and mitogen-activated protein (MAP) kinase activation. In this study, we isolated a human cDNA encoding a novel G-protein-coupled receptor, designated EDG7, and characterized it as a cellular receptor for LPA. The amino acid sequence of the EDG7 protein is 53.7 and 48.8% identical to those of the human functional LPA receptors EDG2 and EDG4, respectively, previously identified. LPA (oleoyl) but not other lysophospholipids induced an increase in the [Ca(2+)](i) of EDG7-overexpressing Sf9 cells. Other LPA receptors, EDG4 but not EDG2, transduced the Ca(2+) response by LPA when expressed in Sf9 cells. LPAs with an unsaturated fatty acid but not with a saturated fatty acid induced an increase in the [Ca(2+)](i) of EDG7-expressing Sf9 cells, whereas LPAs with both saturated and unsaturated fatty acids elicited a Ca(2+) response in Sf9 cells expressing EDG4. In EDG7- or EDG4-expressing Sf9 cells, LPA stimulated forskolin-induced increase in intracellular cAMP levels, which was not observed in EDG2-expressing cells. In PC12 cells, EDG4 but not EDG2 or EDG7 mediated the activation of MAP kinase by LPA. Neither the EDG7- nor EDG4-transduced Ca(2+) response or cAMP accumulation was inhibited by pertussis toxin. In conclusion, the present study demonstrates that EDG7, a new member of the EDG family of G-protein-coupled receptors, is a specific LPA receptor that shows distinct properties from known cloned LPA receptors in ligand specificities, Ca(2+) response, modulation of adenylyl cyclase, and MAP kinase activation.     

Involvement of heparan sulfate proteoglycan in sensing hypotonic stress in bovine aortic endothelial cells

Hypotonic stress (HTS) induces various responses in vascular endothelium, but the molecules involved in sensing HTS are not known. To investigate a possible role of heparan sulfate proteoglycan (HSPG) in sensing HTS, we compared the responses of control bovine aortic endothelial cells (BAECs) with those of cells treated with heparinase III, which exclusively degrades HSPG. Tyrosine phosphorylation of 125 kDa FAK induced by HTS (-30%) in control cells was abolished in heparinase III-treated BAECs. The amplitude of the volume-regulated anion channel (VRAC) current, whose activation is regulated by tyrosine kinase, was significantly reduced by the treatment with heparinase III. Also, HTS-induced ATP release through the VRAC pore and the concomitant Ca(2+) transients were significantly reduced in the heparinase III-treated BAECs. In contrast, exogenously applied ATP evoked similar Ca(2+) transients in both control and heparinase III-treated BAECs. The transient formation of actin stress fibers induced by HTS in control cells was absent in heparinase III-treated BAECs. Lysophosphatidic acid (LPA) also induced FAK phosphorylation, actin reorganization and ATP release in control BAECs, but heparinase III did not affect these LPA-induced responses. We conclude from these observations that HSPG is one of the sensory molecules of hypotonic cell swelling in BAECs.     

Inhibition of voltage-gated Ca(2+) current by PACAP in rat adrenal chromaffin cells.

It is well established that pituitary adenylate cyclase-activating polypeptide (PACAP) can stimulate catecholamine biosynthesis and secretion in adrenal chromaffin cells. Recent studies from this laboratory demonstrated that PACAP pretreatment inhibits nicotine (NIC)-induced intracellular Ca(2+) transients and catecholamine secretion in porcine adrenal chromaffin cells. Mechanistically, this effect is mediated by protein kinase C (PKC), and based on indirect evidence, is thought to primarily target voltage-gated Ca(2+) channels. The present study used whole-cell patch-clamp analysis to test this possibility more directly in rat chromaffin cells. Consistent with the porcine data, pretreatment with PACAP or with phorbol ester [phorbol myristate acetate (PMA)] significantly suppressed NIC-induced intracellular Ca(2+) transients and catecholamine secretion in rat chromaffin cells. Exposure to PACAP and PMA significantly reduced peak Ca(2+) current in rat cells. The effects of both PACAP and PMA on Ca(2+) current could be blocked by treating cells with the PKC inhibitor staurosporine. Exposure to selective channel blockers demonstrated that rat chromaffin cells contain L-, N- and P/Q-type Ca(2+) channels. PACAP pretreatment significantly reduced Ca(2+) current gated through all three channel subtypes. These data suggest that PACAP can negatively modulate NIC-induced catecholamine secretion in both porcine and rat adrenal chromaffin cells.     

Peroxynitrite resistance of sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery endothelium and smooth muscle

We examined the effects of peroxynitrite pre-treatment on sarco/endoplasmic reticulum Ca(2+) (SERCA) pump in pig coronary artery smooth muscle and endothelium. In saponin-permeabilized cells, smooth muscle showed much greater rates of the SERCA Ca(2+) pump-dependent (45)Ca(2+) uptake/mg protein than did the endothelial cells. Peroxynitrite treatment of cells inhibited the SERCA pump more severely in smooth muscle cells than in endothelial cells. To determine implications of this observation, we next examined the effect of the SERCA pump inhibitor cyclopiazonic acid (CPA) on intracellular Ca(2+) concentration of intact cultured cells. CPA produced cytosolic Ca(2+) transients in cultured endothelial and smooth muscle cells. Pre-treatment with peroxynitrite (200 microM) inhibited the Ca(2+) transients in the smooth muscle but not in the endothelial cells. CPA contracts de-endothelialized artery rings and relaxes precontracted arteries with intact endothelium. Peroxynitrite (250 microM) pre-treatment inhibited contraction in the de-endothelialized artery rings, but not the endothelium-dependent relaxation. Thus, endothelial cells appear to be more resistant than smooth muscle to the effects of peroxynitrite at the levels of SERCA pump activity, CPA-induced Ca(2+) transients in cultured cells, and the effects of CPA on contractility. The greater resistance of endothelium to peroxynitrite may play a protective role in pathological conditions such as ischemia-reperfusion when excess free radicals are produced.     

Lysophosphatidic acid-induced Ca2+ mobilization requires intracellular sphingosine 1-phosphate production. Potential involvement of endogenous EDG-4 receptors

Lysophosphatidic acid (LPA)-mediated Ca(2+) mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4, 5-trisphosphate (Ins(1,4,5)P(3))- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca(2+) signaling cascade based upon the lipid second messenger molecule, sphingosine 1-phosphate. Both LPA and direct G-protein activation increase [(3)H]sphingosine 1-phosphate levels in SH-SY5Y cells. Measurements of (45)Ca(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine 1-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca(2+). Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca(2+) "puffs," which resulted from an interaction between the sphingolipid Ca(2+) release pathway and Ins(1,4,5)P(3) receptors. Down-regulation of Ins(1,4,5)P(3) receptors uncovered a Ca(2+) response to LPA, which was manifest as a progressive increase in global cellular Ca(2+) with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine 1-phosphate to stimulate Ca(2+) mobilization in SH-SY5Y cells. Unlike traditional Ca(2+) release processes, this novel pathway does not require the progressive recruitment of elementary Ca(2+) events.     

The influence of lysophosphatidic acid on the functions of human dendritic cells

Lysophosphatidic acid (LPA) is a bioactive lipid mediator which is generated by secretory phospholipase A(2). In this study, we studied the biological activity of LPA on human dendritic cells (DCs), which are specialized APCs characterized by their ability to migrate into target sites and secondary lymphoid organs to process Ags and activate naive T cells. We show that immature and mature DCs express the mRNA for different LPA receptors such as endothelial differentiation gene (EDG)-2, EDG-4, and EDG-7. In immature DCs, LPA stimulated pertussis toxin-sensitive Ca(2+) increase, actin polymerization, and chemotaxis. During the maturation process, DCs lost their ability to respond toward LPA with Ca(2+) transients, actin polymerization, and chemotaxis. However, LPA inhibited in a pertussis toxin-insensitive manner the secretion of IL-12 and TNFalpha as well as enhanced secretion of IL-10 from mature DCs. Moreover, LPA did not affect the endocytic or phagocytic capacities and the surface phenotype of DCs, although it increased the allostimulatory function of mature DC and inhibited their capacity to induce Th1 differentiation. In summary, our study implicates that LPA might regulate the trafficking, cytokine production, and T cell-activating functions of DCs.     

InsP3 receptor type 2 and oscillatory and monophasic Ca2+ transients in rat adrenal chromaffin cells

Muscarinic receptor stimulation induced oscillatory and monophasic Ca(2+) transients in rat adrenal chromaffin cells in the absence of external Ca(2+). As this Ca(2+) mobilization may be mediated by InsP(3), we first explored types of InsP(3) receptors and their intracellular distribution in chromaffin cells. The InsP(3) receptor type 1 was not immunodetected in precipitates of adrenal medulla homogenates and in dissociated adrenal chromaffin cells, whereas an anti-type 3 mAb recognized a faint band with about 250 kDa, but no significant immunoreaction was visible in chromaffin cells. The anti-type 2 mAb strongly detected a band with about 220 kDa and the immunoreaction was observed perinuclearly and at the cell periphery. These results indicate that InsP(3) receptor type 2 is predominant in chromaffin cells. The oscillatory and monophasic Ca(2+) transients were reproduced in simulation based on a three-state kinetic model (shut, open, and inactivated states). Ca(2+) ions were found experimentally and theoretically to turn over rapidly between stores and the cytosol during stimulation. The results suggest that InsP(3) receptor type 2 is responsible for both oscillatory and monophasic Ca(2+) transients and that change in mode of Ca(2+) responses may be accounted for by the kinetic property of the type 2 receptor.     

Development of depolarization-induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K(+) concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K(+) (1 min) with a transient increase in [Ca(2+)](i) of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K(+), the amplitude of the [Ca(2+)](i) transients averaging 592 nM. At later stages, all cells responded to 25 mM K(+) with [Ca(2+)](i) transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca(2+)](i) transients. The amplitudes of these [Ca(2+)](i) transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca(2+)](i) transients were greatly reduced in Ca(2+)-free, EGTA-buffered saline, and in the presence of the Ca(2+) channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca(2+) influx through voltage-activated Ca(2+) channels into antennal lobe glial cells. The development of the depolarization-induced Ca(2+) transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna.     

Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

During early apoptosis, adult cardiomyocytes show unusual beating, suggesting possible participation of abnormal Ca(2+) transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe beta-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca(2+) concentration and cell length. Trains of Ca(2+) transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca(2+) transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca(2+) transients declined and diastolic intracellular Ca(2+) concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca(2+) channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca(2+)-ATPase (thapsigargin), and Na(+)/Ca(2+) exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins (alpha-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca(2+) transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown.