BBA - Molecular Cell Research (v.1496, #2-3)

Many developmental processes are regulated by intercellular signaling mechanisms that employ the activation of receptor tyrosine kinases. One model system that has been particular useful in determining the role of receptor tyrosine kinase-mediated signaling processes in cell fate determination is the developing Drosophila eye. The specification of the R7 photoreceptor cell in each ommatidium of the developing Drosophila eye is dependent on activation of the Sevenless receptor tyrosine kinase. This review will focus on the genetic and biochemical approaches that have identified signaling molecules acting downstream of the Sevenless receptor tyrosine kinase which ultimately trigger differentiation of the R7 photoreceptor cell.

We have investigated the functional coupling of α and β isoforms of the human thromboxane A2 receptor (TP) to Gα16 and Gα12 members of the Gq and G12 families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.α10 or HEK.β3, stably over-expressing TPα and TPβ, respectively. Moreover, using HEK.TPΔ328 cells which over-express a variant of TP truncated at the point of divergence of TPα and TPβ, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPα and TPβ couple similarly to Gα16 to affect increases in inositol 1,4,5-trisphosphate (IP3) and mobilisation of intracellular calcium ([Ca2+]i) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca2+]i mobilisation in cells co-transfected with Gα12, neither receptor generated corresponding increases in IP3, indicating that the Gα12-mediated increases in [Ca2+]i do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca2+ channels, reduced [Ca2+]i mobilisation in TPα and TPβ cells co-transfected with Gα12 to approximately 40% of that mobilised in its absence, whereas [8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca2+ release, had no effect on [Ca2+]i mobilisation by either receptor isoform co-transfected with Gα12. Despite the lack of differential coupling specificity by TPα and TPβ, TPΔ328 signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Gα11, Gα12 or Gα16 subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPα but not TPβ, TPΔ328 coupled to Gαs, leading to increased adenosine 3′,5′-cyclic monophosphate (cAMP), rather than to Gαi. Whereas TPΔ328 signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPα, co-transfection of Gαs did not augment cAMP generation by TPΔ328. Hence, from these studies involving the wild type TPα, TPβ and TPΔ328, we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Gα11 and Gα16 members of the Gq family or to Gα12; it may play a role in determining Gs versus Gi coupling and may act as a determinant of coupling efficiency.
Keywords: Thromboxane A2 receptor; G12; G16; Gs; Signaling;

Effect of N-acetylcysteine and deferoxamine on endogenous antioxidant defense system gene expression in a rat hepatocyte model of cocaine cytotoxicity by Asunción Zaragoza; Carmen Dı́ez-Fernández; Alberto M. Alvarez; David Andrés; Marı́a Cascales (183-195).
In the present study we investigated on cultures of hepatocytes from phenobarbital-pretreated rats, the effect of the antioxidants, 0.5 mM N-acetylcysteine (NAC) or 1.5 mM deferoxamine (DFO), previously incubated for 24 h and coincubated with cocaine (0–1000 μM) for another 24 h. Cocaine cytotoxicity was monitored by either the lysis of the cell membranes or apoptosis. Lysis of the cell membranes was evidenced by lactate dehydrogenase leakage, apoptosis was observed by detecting a hypodiploid peak (<2C) in DNA histograms obtained by flow cytometry, peroxide production was quantified with 2′,7′-dichlorodihydrofluorescein diacetate and gene expression of the antioxidant enzymes: Mn- and Cu,Zn-superoxide dismutases, catalase and glutathione peroxidase were measured by Northern blot analysis. NAC and DFO significantly decreased the extent of lysis of cell membranes and apoptosis, and the antiapoptotic effect was parallel to peroxide generation. By the effect of NAC and DFO, significant increases were detected in the levels of mRNA of catalase, manganese superoxide dismutase and glutathione peroxidase. From these results we conclude that NAC or DFO, when incubated in the presence of cocaine, exerted a protective effect against cocaine toxicity at the level of both lysis of the membranes and apoptosis. This protective effect, in the case of NAC, was directed towards an increase in antioxidant enzyme expression, and in the case of DFO against reactive oxygen species generation.
Keywords: Cocaine cytotoxicity; Oxidative stress; Peroxide; Antioxidant enzyme expression; Apoptosis/necrosis;

The interferon-β and tamoxifen combination induces apoptosis using thioredoxin reductase by Daniel J. Lindner; Edward R. Hofmann; Sreenivasu Karra; Dhananjaya V. Kalvakolanu (196-206).
Interferons (IFNs) suppress cell growth by inducing cellular genes. The anti-estrogen tamoxifen (Tam), binds to estrogen receptor and inhibits transcription of estrogen stimulated genes. In cells resistant to IFN-induced growth suppression, IFN/Tam combination causes cell death. We previously reported that the combination of IFN-β and Tam was a more potent growth suppressor of human tumor xenografts than either agent alone. The IFN/Tam combination acts in a manner similar to the IFN/retinoic acid combination. Using a genetic technique, we have recently identified several genes associated with retinoid-IFN-induced mortality (GRIM). One such gene, GRIM-12, was identical to human thioredoxin reductase (TR). In the present study we have examined whether the IFN/Tam combination also requires GRIM-12 for inducing cell death. We report here that GRIM-12 is necessary for mediating the cell death effects of IFN/Tam, and its expression is induced by IFN/Tam at a post-transcriptional stage. Repression of GRIM-12 levels either by antisense expression or by dominant negative inhibitors caused resistance to IFN/Tam induced death and promoted cell growth. Overexpression of GRIM-12 increased IFN/Tam induced apoptosis. Thus, these studies have identified a critical role for GRIM-12 (TR) in apoptosis.
Keywords: Interferon; Tamoxifen; Thioredoxin reductase; Apoptosis;

Phosphatidylinositol 3-kinase inhibitors, Wortmannin or LY294002, inhibited accumulation of p21 protein after γ-irradiation by stabilization of the protein by Kunihiko Fukuchi; Hiroyuki Watanabe; Shigeru Tomoyasu; Sachiko Ichimura; Kouichi Tatsumi; Kunihide Gomi (207-220).
Expression of the cyclin kinase inhibitor, p21, is regulated both transcriptionally and posttranscriptionally by the ubiquitin-proteasome degradation pathway. Recently, we reported that DNA damage is required for efficient p21 expression by demonstrating that enhanced p21 mRNA expression induced by DNA damage results in increased p21 protein, but enhanced p21 mRNA without DNA damage does not. In addition, we demonstrated that DNA damage suppressed the ubiquitination of p21. In this study, we analyze the link between p21 stabilization and DNA damage. Enhanced p21 protein expression in ML-1 cells resulting from 15 Gy γ-irradiation was diminished by Wortmannin or LY294002 pretreatment of cells. However, the levels of p21 mRNA were not affected by inhibitor pretreatment. Wortmannin or LY294002 pretreatment reduces p53 expression after γ-irradiation to a lesser degree than that of p21. In addition, we examined the involvement of DNA-PK, whose activity is inhibited by Wortmannin or LY294002, in p21 stabilization using the SCID fibroblast cell line and a DNA-PK targeting ML-1 cell line. Accumulation of p21 protein by γ-irradiation was similar to that of DNA-PK intact cells and was reduced by Wortmannin or LY294002 pretreatment. Involvement of another DNA damage detecting enzyme, the ATM gene product, whose activity is also inhibited by Wortmannin or LY294002, was evaluated. ATM deficient cells induced p21 after γ-irradiation, γ-irradiation-induced p21 protein was diminished by pretreatment of cells with Wortmannin or LY294002. We conclude that the p21 stabilization mechanism functions after γ-irradiation, was sensitive to Wortmannin or LY294002, and required neither DNA-PK nor ATM gene product for activity.
Keywords: p21; DNA damage; Wortmannin; Ataxia telangiectasia mutated; DNA dependent protein kinase;

Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells by Katsuya Ikuta; Yoshinori Fujimoto; Yasuaki Suzuki; Kohji Tanaka; Hiroyuki Saito; Motoyuki Ohhira; Katsunori Sasaki; Yutaka Kohgo (221-231).
HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and β2-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the 59Fe uptake and release, and internalization and recycling of 125I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron (59Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4.3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5×105/cell to 1.2×105/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with 125I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.
Keywords: HFE; Hemochromatosis; Transferrin; Recycling; Iron;

Rapid isolation and characterization of CHO mutants deficient in peroxisome biogenesis using the peroxisomal forms of fluorescent proteins by Masaki Ito; Ritsu Ito; Yuan Huang; Satoshi Miura; Atsushi Imamura; Yasuyuki Suzuki; Nobuyuki Shimozawa (232-242).
We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.
Keywords: CHO mutant; Peroxisome assembly; Peroxisome biogenesis disorder; Green fluorescent protein; Blue fluorescent protein;

It was investigated why the fMLP-stimulated respiratory burst in human neutrophils was enhanced by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a considered calmodulin antagonist, at lower concentration but inhibited at higher concentration. Flow cytometric analysis on binding of the receptor to the fluorescence-labeled formyl peptide and the polymerization of actin in cells showed that the drug inhibited actin polymerization and promoted expression of the fMLP receptors on cell membrane at lower concentration, while promoted the actin polymerization and depressed the receptor expression at higher concentration. As intracellular Ca2+ ([Ca2+]i) is elevated, polymerization of actin decreases and the receptor expression increases. At normal physiological and two moderately high intracellular calcium levels, the dual effect of W-7 became less significant as [Ca2+]i was elevated indicating that the dual effect is calcium-dependent. Under two extreme conditions that the intracellular calcium was either depleted or highly elevated, the dual effect disappeared but only an inhibitory effect on actin polymerization was observed. Colchicine and taxol study showed that disruption or stabilization of microtubules had no effect on formyl peptide receptor expression. The results suggest that W-7 primes the fMLP stimulation by direct action on actin leading to breakdown of microfilaments and more expression of formyl peptide receptors, and inhibits the stimulation by indirect action on actin through inactivation of some Ca2+-dependent proteins resulting in assembly of actin into microfilaments. Which action is favorable depends on the drug concentration.
Keywords: W-7; Neutrophil; Respiratory burst; Formyl peptide receptor; Actin polymerization; Calmodulin;

Volume sensitive efflux of taurine in HEK293 cells overexpressing phospholemman by Marcela Morales-Mulia; Herminia Pasantes-Morales; Julio Morán (252-260).
The role of the phospholemman (PLM) on the efflux of taurine and chloride induced by swelling was studied in HEK293 cells overexpressing stable transfected PLM. PLM, a substrate for protein kinases C and A, is a protein that induces an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. In PLM-overexpressing cells the process of RVD was more rapid and efficient (75%) than in control cells (44%). Also, [3H]taurine and 125I efflux induced by hyposmolarity were markedly increased (30–100%) in two subclones of cells overexpressing PLM. This increased efflux was sensitive to the Cl channel blockers DDF, NPPB and DIDS. Acute treatment of control cells with isoproterenol and norepinephrine induced a significant potentiation (50–60%) of [3H]taurine release induced by hyposmolarity. In PLM-overexpressing cells the potentiation by these drugs was higher (100%). Insulin induced also an increase in [3H]taurine release, but only in PLM-overexpressing cells (50%). These results indicate that PLM may play a role in the RVD and that its phosphorylation may have a physiological significance during this process. The mechanisms involved in this process could include the activation of PLM itself as channel or the modulation of other preexisting channels.
Keywords: Phospholemman; Taurine; Chloride; Volume regulation; HEK293 cell; Anion channel;

Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action. It is expressed mainly in the arcuate nucleus where it plays an important role in the hypothalamic control of feeding and energy homeostasis by antagonism of central melanocortin 4 receptors in mammals. Besides in the brain, the melanocortin 4 receptor is expressed in numerous peripheral tissues in the chicken. To examine whether or not the peripheral melanocortin 4 receptor signaling could be regulated by AGRP, we cloned and localized the expression of the AGRP gene in the chicken. The chicken AGRP gene was found to encode a 154 or 165 amino acid protein, depending on the usage of two alternative translation initiation sites. The coding sequence consisted of three exons, like that of mammalian species. The C-terminal cysteine-rich region of the predicted AGRP displayed high levels of identity to mammalian counterparts (78–84%) and all 10 cysteine residues conferring functional conformation of AGRP were conserved; however, other regions showed apparently no homology, suggesting that biological activities of AGRP are located in its C-terminal region. RT-PCR analysis detected the AGRP mRNA in all tissues examined: the brain, adrenal gland, heart, liver, spleen, gonads, kidney, uropygial gland, skeletal muscle and adipose tissues. Interestingly, the skin also expressed the AGRP mRNA, where Agouti, another melanocortin receptor antagonist regulating hair pigmentation, is expressed in rodents. Most of those AGRP-expressing tissues have been demonstrated to express melanocortin 4 receptors and/or other subtypes of melanocortin receptor whose mammalian counterparts can bind AGRP. These results imply the possibility that some peripheral melanocortin systems could be regulated by the functional interaction between melanocortins and AGRP at melanocortin receptors in the chicken.
Keywords: Agouti-related protein; Cloning; Reverse transcription polymerase chain reaction; Tissue distribution; Melanocortin; Chicken;

The effect of gossypol on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca2+ probe. Gossypol evoked a rise in cytosolic free Ca2+ levels ([Ca2+]i) concentration-dependently between 2 and 20 μM. The response was decreased by external Ca2+ removal. In Ca2+-free medium pretreatment with gossypol nearly abolished the [Ca2+]i increase induced by carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, and thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump; but pretreatment with CCCP and thapsigargin only partly inhibited gossypol-induced Ca2+ release. Addition of 3 mM Ca2+ induced a [Ca2+]i increase after pretreatment with 5 μM gossypol in Ca2+-free medium. This Ca2+ entry was decreased by 25 μM econazole, 50 μM SKF96365 and 40 μM aristolochic acid (a phospholipase A2 inhibitor). Pretreatment with aristolochic acid inhibited 5 μM gossypol-induced internal Ca2+ release by 55%, but suppression of phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) had no effect. Gossypol (5 μM) also increased [Ca2+]i in human bladder cancer cells and neutrophils. Collectively, we have found that gossypol increased [Ca2+]i in MDCK cells by releasing Ca2+ from multiple Ca2+ stores in a manner independent of the production of inositol-1,4,5-trisphosphate, followed by Ca2+ influx from external space.
Keywords: Gossypol; Ca2+; Intracellular; Fura-2; MDCK cell; Neutrophil; Bladder carcinoma;

Sodium nitroprusside (SNP) elicits various physiological effects, in part through generation of the membrane permeable mediator nitric oxide (NO). In the heart, besides its role in regulating contractility, NO is involved in both protection from and induction of cellular damage. The present study investigated the role of SNP in the regulation of the mitogen-activated protein kinases (MAPKs) in isolated adult rat cardiomyocytes. SNP maximally activated Erk1, Erk2, p38 MAPK and MAPKAPK2 in 5–10 min. The activation of MAPKAPK2 by SNP was blocked by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolol[4,3-a]quinoxalin-1-one (ODQ) and the p38 MAPK inhibitor, SB203580. The activation of Erk1 was insensitive to ODQ but completely blocked by the Mek1 inhibitor PD98059. The membrane-permeable homologue of cGMP, 8-Br-cGMP, also activated p38 MAPK (A 0.5≈50 μM) but not Erk1 and Erk2. These results indicate that p38 MAPK and MAPKAPK2 are activated by SNP in cGMP-dependent pathways, while the Erk1 activation by SNP is independent of cGMP levels.
Keywords: Sodium nitroprusside; cGMP; Extracellular signal-regulated kinase; p38 Mitogen-activated protein kinase; MAPKAPK2;

Treatment of v-Ki-Ras-transformed SVC1 cells with low retinoic acid induces malignancy reversion associated with Ras p21 down-regulation by Annamaria Spina; Emilio Chiosi; Silvio Naviglio; Mario Pagano; Gennaro Illiano; Magda Marchese; Silvana Russo Spena; Elisabetta Buommino; Francesco Morelli; Salvatore Metafora (285-295).
The effect of nontoxic, low concentrations (10−8 M) of retinoic acid (RA) for a relatively long time (28 days) on a Kirsten ras-virus transformed cell line (Ki-SVC1), derived from the rat seminal vesicle epithelium, was investigated. In these experimental conditions, the cell treatment with RA induced a decrease of the proliferation rate, apoptosis and a marked reduction of both anchorage-independent growth and tumorigenicity. These biological responses were either preceded or associated with important changes in adenylate cyclase/protein kinase C signaling pathways, the activation of important apoptosis-linked genes and a marked decrease of the v-Ki-ras p21 protein. The significance of these findings is discussed.
Keywords: Retinoic acid; Signal transduction pathway; v-Ki-ras p21; Apoptosis; Malignancy reversion;

GRP94 is an inducible resident endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) glycoprotein that functions as a protein chaperone and Ca2+ regulator. GRP94 has been reported to be a substrate for protein kinase CK2 in vitro, although its phosphorylation in intact cells remains unreported. In Sf21 insect cells, overexpression of canine GRP94 led to the appearance of a multiplet of three or more molecular-mass isoforms which was reduced to a single mobility form following treatment of cells with tunicamycin, suggesting stable accumulations of consecutively modified protein. Metabolic labeling of Sf21 cells with 32Pi led to a constitutive phosphorylation of GRP94 which, based upon phosphopeptide mapping, occurred specifically on CK2-sensitive sites. Among the GRP94 multiplet, however, only the lowest mobility form of GRP94 was phosphorylated, even though in vitro phosphorylation of GRP94 by CK2 led to phosphorylation of all glycosylated forms. The 32Pi incorporation into GRP94 indicated a slow turnover of phosphate incorporation that was unaffected by inhibition of biosynthesis, resulting in a steady-state level of phospho-GRP94 on CK2 sites. These data support a role for protein kinase CK2 in the cell biology for GRP94 and other resident ER/SR proteins that may occur in ER compartments.
Keywords: GRP94; Glycosylation; CK2; Phosphorylation; Endoplasmic reticulum;

Previous studies have shown that transforming growth factor-β1 (TGF-β1) stimulates protein kinase C (PKC) via a mechanism that is independent of phospholipase C or tyrosine kinase, but involves a pertussis toxin-sensitive G-protein. Maximal activation occurs at 12 h and requires new gene expression. To understand the signaling pathways involved, resting zone chondrocytes were incubated with TGF-β1 and PKC activity was inhibited with chelerythrine, staurosporine or H-7. [35S]Sulfate incorporation was inhibited, indicating that PKC mediates the effects of TGF-β1 on matrix production. However, there was little, if any, effect on TGF-β1-dependent increases in [3H]thymidine incorporation, and TGF-β1-stimulated alkaline phosphatase was unaffected, indicating that these responses to the growth factor are not regulated via PKC. TGF-β1 caused a dose-dependent increase in prostaglandin E2 (PGE2) production which was further increased by PKC inhibition. The increase was regulated by TGF-β1-dependent effects on phospholipase A2 (PLA2). Activation of PLA2 inhibited TGF-β1 effects on PKC, and inhibition of PLA2 activated TGF-β1-dependent PKC. Exogenous arachidonic acid also inhibited TGF-β1-dependent increases in PKC. The effects of TGF-β1 on PKC involve genomic mechanisms, but not regulation of existing membrane-associated enzyme, since no direct effect of the growth factor on plasma membrane or matrix vesicle PKC was observed. These results support the hypothesis that TGF-β1 modulates its effects on matrix production through PKC, but its effects on alkaline phosphatase are mediated by production of PGE2 and protein kinase A (PKA). Inhibition of PKA also decreases TGF-β1-dependent proliferation. We have previously shown that PGE2 stimulates alkaline phosphatase through its EP2 receptor, whereas EP1 signaling causes a decrease in PKC. Thus, there is cross-talk between the two pathways.
Keywords: Chondrocyte culture; Transforming growth factor-β1; 24,25-(OH)2D3; Protein kinase C; Alkaline phosphatase; Signal transduction;

Short term regulation of cell-cell communication in TM3 Leydig cells by J.M. Cristancho; A.C. Campos de Carvalho; W.A. Varanda (325-332).
Determination of the junctional conductance (g j) in TM3 Leydig cells by the dual whole cell patch clamp technique (DWCPC) shows that coupling undergoes a rapid and irreversible run down. Addition of ATP or cAMP derivatives to the pipette solution has been shown to prevent this phenomenon in several tissues, but this same treatment is unable to inhibit run down in Leydig cells. Because the run down in junctional conductance may pose serious problems to the interpretation of results, we also measured g j by using the double perforated patch clamp technique (DPPT). Access to the cell interior was achieved by adding 200 μg/ml of nystatin to the pipette solution. With this method, run down in g j was greatly reduced, amounting to no more than 5% of the initial value. Exposure of the cells, under DWCPC or DPPT, to dibutyryl cAMP or to tumor promoting agent (TPA) led to a decrease in cell to cell communication. Staurosporine, a PKC inhibitor, increased g j and was able to prevent and reverse the uncoupling action of cAMP or TPA. Our results indicate that cell-cell communication in Leydig cells is down regulated by both protein kinases A and C, interacting in a complex manner.
Keywords: Leydig cell; Gap junction; Protein kinase; Patch clamp; Run down;

Gene expression of flap endonuclease-1 during cell proliferation and differentiation by In-Sook Kim; Mun-Yong Lee; Il-Ha Lee; Soon-Lim Shin; Soo-Young Lee (333-340).
It has been shown that flap endonuclease-1 (FEN-1), a structure-specific nuclease, acts on the removal of RNA primers during Okazaki fragment maturation in DNA synthesis. To study whether the gene expression of FEN-1 is inducible during cell proliferation, we analyzed the FEN-1 mRNA levels in actively growing cells and non-growing cells. The gene expression of FEN-1 was higher in mitotic cells than in resting cells, and was markedly decreased, especially, when terminal differentiation was induced in promyelocytic leukemia cells (HL-60 cells). The decline correlated substantially with the ceasing of DNA synthesis. In the examination of tissue-specific gene expression, the human testis, spleen, thymus and mucosal lining of colon tissues expressed this gene actively, whereas the prostate, ovary, small intestine and peripheral blood leukocyte hardly expressed it. In addition, FEN-1 was co-localized with the proliferating cell nuclear antigen (PCNA) in young rat kidney according to immunohistochemistry. These findings suggest that FEN-1 gene expression is inducible during cell proliferation for DNA synthesis, and is down-regulated during cell differentiation.
Keywords: Cell proliferation; Differentiation; Flap endonuclease-1; Gene expression; Proliferating cell nuclear antigen;

Mitochondrial DNA-depleted neuroblastoma (Rho°) cells exhibit altered calcium signaling by Todd B. Sherer; Patricia A. Trimmer; Janice K. Parks; Jeremy B. Tuttle (341-355).
To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho°). Basal cytosolic calcium levels were slightly but significantly reduced in Rho° cells. The impact of chronic depletion of mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium transients generated in Rho° cells following application of carbachol were more rapid than those in SH-SY5Y cells. A plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho° cells. The rapid calcium transients in Rho° cells were due, in part, to increased reliance on Na+/Ca2+ exchange activity at the plasma membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular calcium stores. Rho° cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen in Rho° cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a consequence of chronic depletion of mitochondrial DNA.
Keywords: Parkinson’s disease; Alzheimer’s disease; Na+/Ca2+ exchange; Mitochondrion; Membrane potential; Calcium influx; Endoplasmic reticulum;

During a yeast two hybrid screen of a Dictyostelium cDNA library using the Ca2+-binding protein CBP1 as bait, we isolated a full-length cDNA encoding a novel Ca2+-binding protein (termed CBP4a). The protein is composed of 162 amino acids and contains four consensus EF-hands. PCR amplification of Dictyostelium genomic DNA using primers specific for the cDNA sequence resulted in the isolation of a gene encoding a different Ca2+-binding protein of 162 amino acids (designated CBP4b) with 90% amino acid sequence identity to CBP4a. Southern blot analysis confirmed the presence of two closely related genes in the Dictyostelium genome. CBP4a and CBP4b mRNAs are expressed at the same stages of development as CBP1 mRNA. In addition, both novel proteins bind 45Ca2+ and interact with CBP1 in vitro in a Ca2+-dependent manner.
Keywords: Ca2+-binding protein; Heterologous expression; Protein-protein interaction; Dictyostelium discoideum;