Shear stress-induced release of basic fibroblast growth factor from endothelial cells is mediated by matrix interaction via integrin alpha(v)beta3

Considering that chronic elevation of shear stress results in remodeling of the vasculature, we analyzed whether mechanical load could mediate basic fibroblast growth factor (bFGF) release and whether bFGF would act as mediator of shear stress-induced endothelial proliferation and differentiation. Supernatant media of shear stress-exposed endothelial cells (EC) contained significantly higher amounts of bFGF than medium from static cells. Released bFGF was fully intact with regard to its function as an inductor of proliferation and differentiation. Shear stress-conditioned media induced capillary-like structure formation, whereas static control medium did not. Likewise, only shear stress-conditioned medium induced proliferation of serum starved EC. Both capillary-like structure formation and proliferation could be inhibited by neutralization of bFGF or its receptor. The release of bFGF was subject to specific, integrin-mediated control, since inhibition of alpha(v)beta(3) integrin prevented it, whereas inhibition of alpha(5)beta(1) integrin had no effect. We conclude that shear stress induces the release of bFGF from EC in a tightly controlled manner. The release is dependent on specific cell-matrix interactions via alpha(v)beta(3) integrins. The effects on cell proliferation and differentiation suggest that release of bFGF is functionally significant and may represent a necessary initial step in adaptive remodeling processes induced by shear stress.     

Production of nitric oxide by mussel (Mytilus galloprovincialis) hemocytes and effect of exogenous nitric oxide on phagocytic functions

In the present study, the ability of mussel (Mytilus galloprovincialis) hemocytes to produce nitric oxide (NO) in response to phorbol myristate acetate (PMA) was determined using the Griess reaction. Significant NO production was found in these cells in response to PMA. This stimulation was reversed in the presence of the NO synthase inhibitor, N(G)-methyl-L-arginine (L-NMMA). Moreover, the effect of the pre-incubation of hemocytes with NO was also determined on phagocytic immune functions of mussel hemocytes using two NO donors, glycerin trinitrate (GTN) and S-nitroso-N-acetyl-penicillamine (SNAP). In the case of GTN, a visible cytotoxic effect of the compound at the higher doses was observed. Those GTN concentrations that did not have a negative effect on hemocyte viability did not produce sufficient NO to significantly alter the chemiluminescent response to zymosan in all cases, nor the ability of hemocytes to phagocytose bacteria (Escherichia coli). SNAP, however, did not affect cell viability at either of the concentrations used and produced NO levels up to 13-fold higher than controls after 2 h of incubation. In this case, NO exogenously produced by SNAP significantly inhibited the chemiluminescent response of mussel hemocytes, whereas it did not have a significant effect on the capability of these cells to phagocytose bacteria.     

Inhibition of vesicular stomatitis virus infection by nitric oxide.

Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV-infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system.     

Inhibitory effect of nitric oxide on the replication of a murine retrovirus in vitro and in vivo.

Nitric oxide (NO) exerts microbicidal effects on a broad spectrum of pathogens, including viruses, but its antiretrovirus properties have not yet been described. The purpose of this study was to determine whether NO inhibits murine Friend leukemia virus (FV) replication in vitro and to what extent NO may play a role in defenses against FV infection in mice. Three NO-generating compounds were studied: 3-morpholino-sydononimine (SIN-1), sodium nitroprusside (SNP), and S-nitroso-N-acetylpenicillamine (SNAP). The effects of these three compounds were compared with those of their controls (SIN-1C, potassium ferricyanide, and N-acetylpenicillamine, respectively), which do not generate NO and with that of sodium nitrite (NaNO2). SIN-1, SNP, and SNAP inhibited FV replication in dunni cells in a concentration-dependent manner. In contrast, no significant inhibitory effect was observed with the three controls or NaNO2. Furthermore, the addition of superoxide dismutase did not alter the inhibitory effect of SIN-1, which is also known to generate superoxide anions. No dunni cell toxicity was observed in the range of concentrations tested. We also assessed the effect of NO produced by activated macrophages on FV replication. Macrophages activated by gamma interferon and lipopolysaccharide inhibited FV replication in a concentration-dependent manner. This inhibition was due in part to NO production, since it was reversed by NG-monomethyl L-arginine, a competitive inhibitor of NO synthase. In vivo administration of NG-nitro-L-arginine methyl ester, a competitive inhibitor of NO synthase, significantly increased the viral load in spleen cells of FV-infected mice. These results suggested that NO may play a role in defenses against the murine Friend leukemia retrovirus.     

Comparison of toxic effects of nitric oxide and peroxynitrite on Uronema marinum (Ciliata: Scuticociliatida)

To discover the effects of nitric oxide (NO) and peroxynitrite on Uronema marinum (a ciliate responsible for systemic scuticociliatosis in cultured olive flounder Paralichthys olivaceus), the dose-dependent inhibitory effect of NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) on the proliferation and survival of U. marinum was investigated. The inhibitory effects of exogenous superoxide dismutase (SOD) and catalase on the toxicity of SIN-1 were also investigated. After 24 h of incubation in the presence of 0.2 mM SNAP, the number of ciliates was not statistically different from that of the controls, whereas incubation in the presence of 0.5 mM SNAP reduced the number of parasites significantly to 59.1% of controls. Concentrations of SNAP higher than 0.5 mM resulted in greater reductions in the number of ciliates, but levels of generated NO far exceeded physiological ranges. The number of viable ciliates incubated for 24 h with 0.2 mM SIN-1 was reduced significantly to 25.0%, and all ciliates were killed by incubation in concentrations above 0.5 mM SIN-1. Although SOD decreased the toxic effect of SIN-1 on U. marinum, protection was not complete and did not improve after increasing the SOD concentration from 50 to 400 U ml(-1). Addition of catalase ranging from 500 to 10000 U ml(-1) completely protected U. marinum from SIN-1 toxicity. Ciliates exposed to catalase alone or catalase plus SIN-1 showed significantly higher and dose-dependent proliferation rates compared to controls. Addition of haemoglobin, ranging from 0.5 to 2.0 mg ml(-1), also protected U. marinum from SIN-1 toxicity, and increased the proliferation rate dose-dependently. In conclusion, resistance of U. marinum to oxidative and nitrative stress may allow this pathogen to withstand the NO- and oxygen-radical-dependent killing mechanisms of phagocytic cells.     

Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxin-induced nitric oxide

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-beta prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-alpha plus IFN-gamma are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.     

Nitric oxide (NO) pretreatment increases cytokine-induced NO production in cultured rat hepatocytes by suppressing GTP cyclohydrolase I feedback inhibitory protein level and promoting inducible NO synthase dimerization

Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.     

Differential cytostatic effects of NO donors and NO producing cells.

A 3-h exposure to NO donors (spermine-NO, DETA-NO, or SNAP), or to NOS II-expressing cells (activated macrophages or EMT6 cells) reversibly inhibited DNA synthesis in K562 tumor cells. In GSH-depleted K562 cells, cytostasis remained reversible when induced by DETA-NO or NOS II activity, but became irreversible after exposure to spermine-NO or SNAP. Only SNAP and spermine-NO efficiently inhibited GAPDH, an enzyme with a critical thiol, in GSH-depleted cells. Thus, the irreversible cytostasis induced in GSH-depleted cells by spermine-NO or SNAP can be tentatively attributed to S-nitrosating or oxidizing species derived from NO. However, these species did not contribute significantly to the early antiproliferative effects of macrophages. Ribonucleotide reductase, a key enzyme in DNA synthesis. has been shown to be inhibited by NO. Supplementation of the medium with deoxyribonucleosides to bypass RNR inhibition restored DNA synthesis in target cells exposed to DETA-NO and NO-producing cells, but was inefficient for GSH-depleted cells previously submitted to spermine-NO or SNAP. These cells also exhibited a persistent depletion of the dATP pool. In conclusion, GSH depletion reveals striking qualitative differences in the nature of the toxic effectors released by various NO sources, questioning the significance of S-nitrosating or oxidizing nitrogen oxides in NOS II-dependent cytostasis.     

Nitric oxide modifies glycolytic pathways in cultured human synoviocytes

Nitric oxide (NO) is a free radical produced during inflammation following activation of an inducible NO synthase by pro-inflammatory cytokines such as IL-1beta. Since both NO and IL-1beta are involved in the physiopathology of inflammatory arthropathies, we investigated the effects of exogenous NO on glycolytic pathways in cultured human osteoarthritic synovial cells. NO generated from S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP) inhibited glucose uptake (by 50% after 1 h of incubation) and lactate production by 16% (SNAP) and 8.5% (SNP) after 3 h. Both NO donors also reduced production of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme of the glycolytic pathway. This effect was reversed by haemoglobin, a NO scavenger with higher affinity for the radical. In contrast, the effect on glucose uptake appeared to be irreversible.     

Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells

Vascular endothelial cells (ECs) seeded sparsely on extracellular matrix (ECM) will proliferate in the absence of exogenous basic fibroblast growth factor (bFGF). This ECM will also stimulate neurite outgrowth in PC12 cells in the absence of exogenous growth factors. We have previously shown that bFGF is found in subendothelial ECM (Vlodavsky, I., J. Folkman, R. Sullivan, R. Fridman, R. Ishai-Michaeli, J. Sasse, and M. Klagsburn. 1987. Proc. Natl. Acad. Sci. USA. 84:2292-2296) and in basement membranes (Folkman, J., M. Klagsburn, J. Sasse, M. Wadzinski, D. Ingber, and I. Vlodavsky. 1988. Am. J. Pathol. 130:393-400). The actual requirement of ECM-associated bFGF for the growth of ECs and differentiation of PC12 cells was shown in two ways. First, polyclonal anti-bFGF antibodies added to subendothelial ECM inhibited both EC proliferation and PC12 neurite outgrowth. Secondly, PF-HR-9 cells, which do not synthesize bFGF and which produce an ECM not permissive for EC proliferation and PC12 neurite outgrowth, were transfected with bFGF cDNA. PF-HR-9 cells transfected with bFGF, but not with the dominant selectable marker SV2-neomycin, were found to express bFGF and to produce an ECM which did support both EC proliferation and PC12 differentiation. The ECM-mediated stimulatory effects were inhibited by anti-bFGF antibodies but not by anti-nerve growth factor antibodies or nonimmune rabbit IgG. These results indicate that bFGF associated with ECM is a required ECM component for ECM-mediated cell proliferation and differentiation.     

L-NAME pre-treatment partially inhibits the agmatine-evoked depression of the electrically induced twitch contraction of isolated rat vas deferens

The effect of the putative endogenous ligand for alpha(2)-adrenoceptors and imidazoline receptors agmatine was studied in sympathetic neurotransmission in the rat epididymal vas deferens. Tissues were obtained from N(varpi)-nitro-l-arginine methyl ester (l-NAME)-treated or normal animals and were contracted by electrical stimulation or by exogenous adenosine 5'-triphosphate (ATP). In the electrically stimulated epididymal end, agmatine produced an inhibitory effect on twitch contraction that was partially reversed in l-NAME-treated animals, whereas the inhibition produced by clonidine was not affected by l-NAME treatment. The nitric oxide (NO)-donor S-nitroso-N-acetyl-penicillamine (SNAP) also inhibited twitch contraction. Neither agmatine nor SNAP interfered with the responses induced by exogenous ATP in the epididymal end. Removal of the epithelium of the preparation did not modify the agmatine response. We conclude that a nitrergic pathway activated by agmatine plays a role in its inhibitory effect in rat vas deferens, but it remains to be investigated whether it results from a direct action on the enzyme NO-synthase or a receptor-mediated mechanism.     

Nitric oxide and platelet energy metabolism

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.     

Nitric oxide can function as either a killer molecule or an antiapoptotic effector in cardiomyocytes.

Caspase enzymes are a family of cysteine proteases that play a central role in apoptosis. Recently, it has been demonstrated that caspases can be S-nitrosylated and inhibited by nitric oxide (NO). The present report shows that in chick embryo heart cells (CEHC), NO donor molecules such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione, spermine-NO or sodium nitroprusside inhibit caspase activity in both basal and staurosporine-treated cells. However, the inhibitory effect of NO donors on caspase activity is accompanied by a parallel cytotoxic effect, that precludes NO to exert its antiapoptotic capability. N-Acetylcysteine (NAC) at a concentration of 10 mM blocks depletion of cellular glutathione and cell death in SNAP-treated CEHC, but it poorly affects the ability of SNAP to inhibit caspase activity. Consequently, in the presence of NAC, SNAP attenuates not only caspase activity but also cell death of staurosporine-treated CEHC. These data show that changes in the redox environment may inhibit NO-mediated toxicity, without affecting the antiapoptotic capability of NO, mediated by inhibition of caspase enzymes. NO may thus be transformed from a killer molecule into an antiapoptotic agent.     

Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, stimulate myoblast proliferation in vitro

Summary Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1–10 μM) of donor concentrations. However, 50 μM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 μM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.     

Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation.

Several nitric oxide (NO) effects in the cardiovascular system are mediated by soluble guanylate cyclase (sGC) activation but potassium channels (KC) are also emerging as important effectors of NO actions. We investigated the relationship among vascular smooth muscle cell proliferation, NO, cyclic GMP, and KC using the A7r5 smooth muscle cell line derived from rat aorta. NO donors (two nitrosothiols, S-nitroso-acetyl-d,l-penicillamine, SNAP, and S-nitroso-glutathione, GSNO, and an organic nitrate, glyceryl trinitrate, GTN; 1-1000 microM) dose-dependently inhibited cell proliferation. ODQ (a selective inhibitor of sGC; 0.1 and 1 microM) and KT5823 (a selective inhibitor of cGMP-dependent protein kinase, 1 microM) prevented NO effects, confirming that sGC is a key target. In this report, we show that tetraethylammonium (TEA, a non-selective blocker of KC, 300 microM), and 4-aminopyridine (a selective blocker of voltage-dependent KC, 100 microM) prevented SNAP inhibitory effects on cell proliferation, whereas glibenclamide (a selective blocker of ATP-dependent KC, 1 microM) was ineffective. Iberiotoxin (a selective blocker of high conductance calcium-activated KC, 100 nM), as well charybdotoxin (a blocker of high and intermediate conductance calcium-activated KC, 100 nM) and apamine (a selective blocker of small conductance calcium-activated KC, 100 nM), blocked the antiproliferative effect induced by SNAP. NS1619 (an opener of high conductance calcium-activated KC, 1-100 microM), inhibited cell proliferation. In addition, sub-effective concentrations of ODQ (100 nM) and TEA (10 microM) synergized in blocking SNAP antiproliferative effects. Thus, voltage-dependent and calcium-activated but not ATP-dependent KC appear to have a prominent role, besides sGC activation, in NO-induced inhibition of vascular smooth muscle cell proliferation.     

L-arginine inhibits apoptosis via a NO-dependent mechanism in Nb2 lymphoma cells

Prolactin (PRL) inhibits apoptosis and stimulates proliferation of the PRL-dependent rat Nb2 lymphoma cell line by divergent signaling pathways. Nitric oxide (NO) was recently identified as a downstream regulator of PRL action, and as an inhibitor of apoptosis in immune cells. In the present study, the role of NO in PRL-regulated Nb2 cell function was investigated. Nb2 cells expressed the endothelial nitric oxide synthase (eNOS) isoform, whereas neuronal NOS (nNOS) and inducible NOS (iNOS) mRNAs were undetectable. The eNOS mRNA was abundantly expressed in PRL-deprived, growth-arrested cells but decreased by at least 3-fold at 3-24 h following PRL treatment. Downregulation of eNOS was not accompanied by a corresponding decrease in the eNOS protein, the level of which remained constant for at least 24 h after PRL treatment. PRL had no effect on the phosphorylation state or subcellular redistribution of the eNOS enzyme, or on production of NO by Nb2 cells. However, increasing concentrations of L-arginine (NOS substrate) alone increased NO production in these cells and significantly enhanced PRL-stimulated cell proliferation. NO releasers (SNAP, DEA/NO, SIN-1) also significantly enhanced Nb2 cell proliferation in the presence of a submaximal dose of PRL (0.125 ng/ml). In the absence of PRL, the NO releasers alone promoted cell survival and maintained a viable cell density significantly higher than that of untreated PRL-deprived cells. L-arginine or the NO releaser DEA/NO alone significantly inhibited apoptosis in Nb2 cells deprived of PRL for 5 days. Expression of the anti-apoptotic gene bcl-2, which was stimulated within 1 h by PRL, was upregulated by L-arginine or DEA/NO alone at 2 h and 8 h, respectively. These findings suggest that NO produced by eNOS inhibits apoptosis and promotes the survival of growth-arrested Nb2 lymphoma cells via a prolactin-independent, Bcl-2-mediated pathway.     

The involvement of HAb18G/CD147 in regulation of store-operated calcium entry and metastasis of human hepatoma cells

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca(2+) mobilization and metastatic process of human hepatoma cells. HAb18G/CD147 cDNA was transfected into human 7721 hepatoma cells to obtain a cell line stably expressing HAb18G/CD147, T7721, as demonstrated by Northern blot and immunocytochemical studies. 8-Bromo-cGMP (cGMP) inhibited the thapsigargin-induced Ca(2+) entry in a concentration-dependent manner in 7721 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 microm). However, expression of HAb18G/CD147 in T7721 cells decreased the inhibitory response to cGMP. A similar concentration-dependent inhibitory effect on the Ca(2+) entry was observed in 7721 cells in response to a NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca(2+) entry was significantly reduced in HAb18G/CD147-expressing T7721 cells, indicating a role for HAb18G/CD147 in NO/cGMP-regulated Ca(2+) entry. Experiments investigating metastatic potentials demonstrated that HAb18G/CD147-expressing T7721 cells attached to the Matrigel-coated culture plates and invaded through Matrigel-coated permeable filters at the rate significantly greater than that observed in 7721 cells. Both the attachment and invasion rates could be suppressed by SNAP, and the inhibitory effect of SNAP could be reversed by NO inhibitor, N(G)-nitro-l-arginine methyl ester. The sensitivity of the attachment and invasion rates to cGMP was significantly reduced in T7721 cells as compared with 7721 cells when cells were pretreated with thapsigargin. The difference in the sensitivity between the two cells could be abolished by a Ca(2+) channel blocker, Ni(2+) (3 mm). These results suggest that HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca(2+) entry by NO/cGMP.     

Production of nitric oxide by sperm of the steelhead (Oncorhynchus mykiss) and its actions on motility and respiration

The objective was to characterize nitric oxide (NO) involvement in steelhead sperm physiology with respect to modulation of motility and quiescent sperm respiration, and to assess NO production. Activation of sperm motility in the presence of a NO scavenger (PTIO) decreased path straightness (STR; from 62 to 44%, P < 0.05) and wobble (indicator of lateral head movement, WOB; from 68 to 61%, P < 0.05), whereas activating solution containing a NO donor (SNAP) increased STR (from 62 to 71%, P < 0.05). Neither SNAP nor PTIO impacted percent motility or velocity when present in activating media alone. Incubation of quiescent sperm with SNAP reduced motility (from 96 to 53%, P < 0.0001), curvilinear velocity (from 156 to 83 μm/s P < 0.0001), and WOB (from 77 to 50%, P < 0.0001); however, these effects were abolished by inclusion of PTIO. Response of quiescent sperm to SNAP was reversible with time, whereas PTIO alone had no effect. Incubation of sperm with SNAP decreased respiration to approximately one half of control (P < 0.05). With the fluorescent NO indicator, DAF-FM DA, intracellular NO was detected in quiescent, but not activated, sperm. Incubation of activated sperm in an immobilization buffer resulted in reappearance of NO. In addition to illustrating NO sensitivity of steelhead sperm motility, we inferred that the effects of NO on quiescent sperm occurred via inhibition of respiration, and that these sperm produced NO prior to activation.     

Autocrinological role of basic fibroblast growth factor on tube formation of vascular endothelial cells in vitro.

When bovine capillary endothelial (BCE) cells plated on type I collagen gel were covered with a second layer of collage gel, BCE cells reorganized into a network of capillary-like structures. In the presence of affinity purified anti-basic fibroblast growth factor (bFGF) antibody, this reorganization was inhibited. By using a computerized image analyzer, the formation of network structures and the effect of anti-bFGF antibody was quantitated. The inhibitory effect of anti-FGF antibody was dose-dependent and maximal inhibition was observed at 2.0 micrograms/ml of antibody. Exogenously added bFGF potentiated network formation of BCE cells and coadministration of bFGF abrogated the inhibitory effect of anti-bFGF antibody. Platelet factor 4, which blocks the binding of bFGF to its receptor, inhibited network formation. These results indicate that bFGF produced by endothelial cells regulates angiogenesis as an autocrine factor.