INSULIN PRODUCES A BIPHASIC RESPONSE INTETRAHYMENA THERMOPHILABY STIMULATING CELL SURVIVAL AND ACTIVATING PROLIFERATION IN TWO SEPARATE CONCENTRATION INTERVALS

Cells ofTetrahymenamay produce autocrine signal molecules with effects on survival and proliferation. Here we have tested the effects of human recombinant and bovine insulin, and the B22–B30 fragment of bovine insulin over a wide range of concentrations (10^?5–10^?18m ) on cell survival and proliferation in a synthetic nutrient medium. The cells were grown in conical flasks at low initial cell densities (40 and 400cells/ml). Insulin prevented rapid cell death and/or promoted cell proliferation over two separate concentration ranges: down to nanomolar levels and again in the low pico- and femtomolar range. At an initial population density of 400cells/ml the cells multiplied at both concentration intervals. At 40 or fewer organisms/ml the cells multiplied in the high concentration interval, whereas in the low interval they survived for about four times longer than those in the control cultures. B22–B30 added to cultures of 40 initial cells/ml produced a stimulation of cell survival in the low pico- and high femtomolar range. In the presence of hemin (50nm ) cells at 400 initial organisms/ml multiplied at insulin concentrations down to about 3nm and again from 300am to 10pm . In some cases, hemin plus insulin activated cell proliferation between the two concentration intervals as well. At 40cells/ml the cells not only survived but proliferated in the femtomolar range. Cells in cultures supplemented with both hemin and B22–B30 multiplied at the low concentration interval (from about 100fm to 10pm ).     

Inhibition of growth hormone-induced lipolysis by 3',5'-guanosine monophosphate in chicken adipose tissue in vitro

The influence of cyclic 3',5'-guanosine monophosphate (cGMP) on the lipolytic and antilipolytic (inhibition of glucagon-stimulated lipolysis) responses to GH (1 microgram/ml) was examined in chicken adipose tissue in vitro. Both 8-bromo-cGMP (0.1 mM) and sodium nitroprusside (1 mM) (a guanyl cyclase stimulator) completely inhibited the lipolytic effect of GH. A cGMP-lowering agent, LY83583 (10 microM), reversed the inhibitory effect of sodium nitroprusside on GH-stimulated lipolysis. Furthermore, the suppressive effects of insulin (100 ng/ml), insulin-like growth factor I (IGF-I) (100 ng/ml), or insulin-like growth factor II (IGF-II/MSA) (100 ng/ml), but not somatostatin (1 ng/ml), on GH-stimulated lipolysis were prevented by LY83583 addition. Neither 8-bromo-cGMP, sodium nitroprusside, nor LY83583 altered GH-induced inhibition of glucagon (1 ng/ml)-stimulated lipolysis. It is proposed that cGMP may mediate inhibitory control of GH-stimulated lipolysis by insulin, IGF-I, and IGF-II in chicken adipose tissue.     

Nitric oxide priming protects nitric oxide-mediated apoptosis via heme oxygenase-1 induction

The role of nitric oxide (NO) as a cytotoxic effector molecule of the immune system is clearly established, but recent studies demonstrate cytoprotective functions of NO at low nontoxic concentrations. However, the mechanism of cytoprotection has not been defined completely. Thus, we investigate the involvement of heme oxygenase-1 (HO-1) in the cytoprotective effects of NO. Exposure of L929 cells to sodium nitroprusside (SNP) resulted in the induction of HO-1 protein expression and heme oxygenase activity. Pretreatment of the cells with a low dose of NO (200 microM SNP) significantly inhibited a high dose of (1000 microM SNP) NO-induced apoptosis in L929 cells. Cytoprotection by a low dose of NO was abrogated in the presence of the heme oxygenase inhibitor zinc protoporphyrin IX. A cytoprotective effect comparable to a low dose of SNP was observed when the cells were transfected with HO-1 gene or preincubated with another HO-1 inducer, hemin. Additional experiments revealed the involvement of carbon monoxide in the cytoprotective effect of SNP/HO-1 in L929 cells. Our results presented here provide evidence to support the essential role of HO-1 in the cytoprotective function of NO priming.     

Requirement for bivalent cations in the actions of insulin and sodium nitroprusside on metabolism in rat adipocytes

The requirement for Ca2+ and Mg2+ in the actions of insulin and sodium nitroprusside on rat adipocyte metabolism was investigated: sodium nitroprusside, but not insulin, increased cGMP levels in cells incubated in the absence of Ca2+ and/or Mg2+; sodium nitroprusside and insulin are unable to increase the incorporation of [14C]glucose into triglycerides and [14C]leucine into proteins in the absence of Ca2+ and Mg2+; sodium nitroprusside and insulin showed antilipolytic actions in Ca2+- and Mg2+-free medium. We conclude that in the absence of Ca2+ and Mg2+, sodium nitroprusside and insulin have very similar regulatory properties on triglyceride, protein synthesis and adrenaline-stimulated lipolysis, but not on cGMP levels in rat adipocytes. This could provide evidence that omission of bivalent cations was inhibitory at more than one site, or that sodium nitroprusside mimics insulin's actions by another mechanism that does not involve cGMP.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Regulation of transferrin receptors in human hematopoietic cell lines

Cells grown in the presence of ferric ammonium citrate or hemin exhibited a concentration and time-dependent decrease in 125I-transferrin (Trf) binding. In contrast, cells grown in the presence of protoporphyrin IX or picolinic acid (an iron chelator) exhibited a marked increase in Trf binding. The decrease or increase in binding activity observed under these different conditions of culture reflected, respectively, a reduction or increase in receptor number rather than an alteration in ligand receptor affinity. Growth of the cells in the presence of saturating concentrations of apotransferrin only induced a slight reduction in receptor number. Investigation of the Trf receptors' turnover and biosynthesis clearly showed that iron and hemin decreased the synthesis of Trf receptors without any modification of the receptor turnover; in contrast, protoporphyrin IX and picolinic acid markedly increased the synthesis of Trf receptors. Our results suggest that hemin, iron, and protoporphyrin IX may represent the main molecules involved in the regulation of Trf receptors.     

Porphyrin binding by the surface array virulence protein of Aeromonas salmonicida.

Congo red binding by virulent A-layer-containing (A+) and avirulent A-layer-deficient (A-) strains of Aeromonas salmonicida was examined. Congo red binding to A+ cells was enhanced by salt and thus hydrophobically driven, but at low Congo red concentrations binding was salt independent. Congo red was bound by A+ cells by a kinetically distinct mechanism (Kd, 0.25 microM) which was absent in A- isogenic strains. Purified A-layer protein ("A protein") protein A also bound Congo red with similar affinity (Kd, 0.40 microM). Congo red binding was structurally specific; it was not influenced by a wide variety of compounds including amino acids and nucleotides and only weakly inhibited by structurally similar dyes. However, protoporphyrin IX and hemin were strong competitive inhibitors of Congo red binding. Protoporphyrin and hemin were bound only by A+ strains (KdS of 0.41 and 0.63 microM, respectively). Furthermore, binding of these porphyrins was strongly inhibited by Congo red but weakly inhibited by hematoporphyrin. Purified A protein also bound protoporphyrin IX and hemin with affinities similar to those of A+ cells (KdS of 0.94 and 0.41 microM, respectively.     

Regulation of soluble guanylate cyclase activity by porphyrins and metalloporphyrins

Alterations of the chemical structure of protoporphyrin IX markedly altered the activation of soluble guanylate cyclase purified from bovine lung. Hydrophobic side chains at positions 2 and 4 and vicinal propionic acid residues at positions 6 and 7 of the porphyrin ring (protoporphyrin IX, mesoporphyrin IX) were essential for maximal enzyme activation (Ka = 7-8 nM; Vmax = 6-8 mumol of cGMP/min/mg). Substitution of hydrophobic with polar groups (hematoporphyrin IX, coproporphyrin III), or with hydrogen atoms ( deuteroporphyrin IX), and methylation of propionate residues resulted in decreased enzyme stimulation. Stimulatory porphyrins increased the Vmax and the apparent affinities of enzyme for MgGTP and uncomplexed Mg2+. An open central core in the porphyrin ring was essential for enzyme activation. The pyrrolic nitrogen adduct, N-phenylprotoporphyrin IX, was inhibitory and competitive with protoporphyrin IX (KI = 73 nM). Similarly, metalloporphyrins inhibited enzymatic activity and ferro-protoporphyrin IX (KI = 350 nM), zinc-protoporphyrin IX (KI = 50 nM) and manganese-protoporphyrin IX (KI = 9 nM) were competitive with protoporphyrin IX. Inhibitory porphyrins and metalloporphyrins also prevented enzyme activation by S-nitroso-N- acetylpenicillamine and NO. Guanylate cyclase reconstituted with such porphyrins required higher concentrations of protoporphyrin IX for further activation and were not activated by NO. Thus, porphyrins, metalloporphyrins, and NO appeared to interact at a common binding site on guanylate cyclase. This common site is likely that which normally binds heme and, therefore, NO-heme when the heme-containing enzyme is exposed to NO. Thus, NO and nitroso compounds may react with enzyme-bound heme to generate a modified porphyrin which structurally resembles protoporphyrin IX in its interaction with guanylate cyclase.     

Vasodilator-stimulated protein phosphorylation in platelets is mediated by cAMP- and cGMP-dependent protein kinases

Vasodilators such as sodium nitroprusside, nitroglycerin and various prostaglandins are capable of inhibiting platelet aggregation associated with an increase of either cGMP or cAMP. In our studies with intact platelets, prostaglandin E1 and sodium nitroprusside stimulated the phosphorylation of several proteins which could be distinguished from proteins known to be phosphorylated by a calmodulin-regulated protein kinase or by protein kinase C. Prostaglandin E1 (10 microM) or dibutyryl cAMP (2 mM) stimulated the phosphorylation of proteins with apparent relative molecular masses, Mr, of 240,000, 68,000, 50,000, and 22,000 in intact platelets. These proteins were also phosphorylated in response to low concentrations (1-2 microM) of cAMP in a particulate fraction of platelets. In intact platelets, sodium nitroprusside (100 microM) and the 8-bromo derivative of cGMP (2 mM) increased the phosphorylation of one protein of Mr 50,000 which was also phosphorylated in response to low concentrations (1-2 microM) of cGMP in platelet membranes. An additional protein (Mr 24,000) appeared to be phosphorylated to a lesser degree in intact platelets by prostaglandin E1 and sodium nitroprusside. Since the phosphorylation of the protein of Mr 50,000 was stimulated both in intact platelets by cyclic-nucleotide-elevating agents and cyclic nucleotide analogs, as well as in platelet membranes by cyclic nucleotides, this phosphoprotein was analyzed by limited proteolysis, tryptic fingerprinting and phosphoamino acid analysis. These experiments indicated that the 50-kDa proteins phosphorylated by sodium nitroprusside and prostaglandin E1 were identical, and that the peptide of the 50-kDa protein phosphorylated by both agents was also the same as the peptide derived from the 50-kDa protein phosphorylated in platelet membranes by cGMP- and cAMP-dependent protein kinases, respectively. Regulation of protein phosphorylation mediated by cAMP- and cGMP-dependent protein kinases may be the molecular mechanism by which those vasodilators, capable of increasing either cAMP or cGMP, inhibit platelet aggregation.     

Neurotrophic activity of neudesin, a novel extracellular heme-binding protein, is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain

Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme-binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b(5)-like heme/steroid-binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402 nm. The addition of exogenous hemin extensively increased the amount of hemin-bound neudesin. In contrast, neudesinDeltaHBD, a mutant lacking the heme-binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin-hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The neurotrophic activity of neudesin was enhanced by the binding of Fe(III)-protoporphyrin IX, but neither Fe(II)-protoporphyrin IX nor protoporphyrin IX alone. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti-neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme-affinity column at all. Neudesin is the first extracellular heme-binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme-binding proteins.     

Effects of cytochalasin B on cell division and vacuole formation in Tetrahymena pyriformis GL

Cytochalasin-B (Cyt-B) was tested for its effect on cell division and vacuole formation in Tetrahymena. Little effect was found on cell division in the synchronous cell system in concentrations up to 37 μg/ml; however, slight delay was caused by 71 μg/ml. As measured by particle uptake, much lower concentrations, 7–8 μg/ml, caused significant inhibition of vacuole formation in exponentially multiplying and in starved cells, 16.6 or 37 μg/ml caused strong inhibition. This effect was immediate and completely reversible. The presence of Cyt-B caused starvation of Tetrahymena. The essential absence of inhibition of cell division by Cyt-B may reflect that the drug can enter the cell only by way of vacuoles.     

The effects of follicle-stimulating hormone and cyclic guanosine 3',5'-monophosphate on cyclic adenosine 3',5'-monophosphate-phosphodiesterase and resumption of meiosis in hamster cumulus-oocyte complexes

The effects of follicle-stimulating hormone (FSH) and cyclic guanosine 3',5'-monophosphate (cGMP) on spontaneous oocyte maturation and cyclic adenosine 3',5'-cumulus-monophosphate phosphodiesterase activity (cAMP-PDE) were evaluated by using cumulus-oocyte complexes (COCs) from proestrous hamsters. After a 2-h incubation period, FSH (10 micrograms/ml and 1 microgram/ml) reduced the percentage of maturing oocytes compared with controls. This inhibition was partially overcome when cGMP-elevating agents (8-Bromo-cGMP, atrial natriuretic factor or sodium nitroprusside) were included with FSH. After a 3-h period, incubation with FSH and cGMP-elevating agents alone increased the maturation rate above that of the controls. The accelerating effects of cGMP on the maturation rate appear to be caused by its capacity to lower cAMP levels. Combining FSH (1 microgram/ml) with sodium nitroprusside reduced cAMP levels in COCs (not oocytes) compared with groups exposed to FSH alone. FSH increased cGMP levels in COCs in a dose- and time-dependent manner. Both FSH and cGMP-elevating agents produced a dose-dependent increased cAMP-PDE activity in COCs (not oocytes) following a 2-h incubation period. Together, these results suggest that, in vivo, FSH stimulates a rise in both cAMP and cGMP in COCs. While the increase in cAMP may be the initial meiotic trigger, cGMP may serve to subsequently lower cAMP by activating cAMP-PDE and thus permit the maturational process to continue.     

Biosynthesis of delta-aminolevulinic acid and the regulation of heme formation by immature erythroid cells in man

Heme formation in the erythron is subject to end product regulation by negative feedback, but the exact point of metabolic control in human erythroid cells is unknown. To investigate the mode of action of heme on its own formation, the effects of micromolar concentrations of hemin on de novo synthesis of protoporphyrin IX and delta-aminolevulinate (delta-ALA) by intact human reticulocytes were examined in the presence of 1 mM alpha,alpha'-bipyridyl and 200 microM 4,6-dioxoheptanoate to block their further conversion by ferrochelatase or delta-ALA dehydrase, respectively. At final concentrations (25-40 microM), hemin, which is known to reduce incorporation of [2-14C]glycine into cellular heme, significantly inhibited formation of protoporphyrin IX and total delta-aminolevulinate in situ by these cells. Since synthesis of the first committed precursor, delta-aminolevulinate, as well as protoporphyrin (which is derived from it) were diminished, the effects of hemin on delta-aminolevulinate synthase (EC 2.3.1.37) were studied. Hemin, at concentrations up to 40 microM, had no direct effect on enzymatic activity, as measured with [5-14C] alpha-ketoglutarate (in hypotonically lysed cells) or [1,4-14C]succinyl coenzyme A (in deoxycholate lysates), even after preincubation. However, when intact human reticulocytes were incubated with hemin before assay for delta-ALA synthase, there was a rapid, concentration-dependent reduction in enzymatic activity (mean 42 and 23% inhibition after 60 min for these two substrates, respectively). Hemin had no effect on steady-state levels of delta-ALA synthase mRNA, as determined by Northern blot hybridization using an erythroid-specific human cDNA probe. Thus, a mechanism for inducing feedback inhibition of the tetrapyrrole pathway exists in human erythroid cells. It controls formation of the first committed precursor of protoporphyrin IX, delta-aminolevulinate, and hence regulates heme biosynthesis by limiting the availability of the porphyrin, rather than the metal substrate for the ferrochelatase reaction. Hemin interacts with constituents of the intact reticulocyte significantly to reduce delta-aminolevulinic acid synthase activity by an indirect cellular process that does not influence the abundance of erythroid-specific synthase mRNA but may either inhibit its ribosomal translation in an unknown manner or promote degradation of the enzyme itself by specific proteolysis.     

The use of <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis> mutant cell line for removal of cholesterol from milk

The nonpathogenic ciliate Tetrahymena thermophila converts cholesterol from foodstuffs into provitamin D compounds in high yields. However, prolonged incubation with wild-type strain CU-399 at high densities results in a final deterioration of milk properties, possibly as a result of secreted hydrolases. Here we attempted to solve this problem using MS-1 Tetrahymena strain, a stable mutant with a low rate of hydrolase secretion. Densities of to 2 × 10 ⁶ cells/ml can be incubated for up to 5 h in milk, without any clotting or change in appearance. Moreover, centrifugation of this suspension eliminates most of the cells, and results in an about 75% ± 10 (n = 10) decrease of the initial cholesterol. Sterols are recovered in the cell pellets, which show that Tetrahymena is able to avidly capture them from the medium. Therefore, this mutant strain is optimal for milk cholesterol depletion, avoiding unfavorable sensory alterations.     

Release of tumor necrosis factor-alpha from macrophages. Enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides

PGE2 has previously been shown to suppress various leukocyte functions. In this study, we examined whether PGE2 would affect release of TNF-alpha from rat resident peritoneal macrophages. Two different, dose-dependent effects were observed: low PGE2 concentrations (0.1 to 10 ng/ml) stimulated, whereas higher concentrations (greater than 10 ng/ml) suppressed TNF-alpha release. PGE2-stimulated TNF-alpha production was dependent on de novo protein synthesis and was associated with an intracellular rise of cGMP. The importance of cGMP as an intracellular messenger for PGE2 was confirmed by the following evidence: (1) low PGE2 concentrations preferentially increased cGMP and not cAMP and (2) cGMP, either exogenously added or endogenously generated by sodium nitroprusside, were efficient stimulators of TNF-alpha production. In contrast, agents increasing intracellular cAMP concentrations such as PGE1, higher PGE2 doses, isoproterenol, and theophylline, all suppressed TNF-alpha synthesis. Only resident, but not casein-elicited or Corynebacterium parvum-activated macrophages, were stimulated by low PGE2 concentrations to increase TNF-alpha production. In tumor cytotoxicity assays, PGE2-activated macrophages were active only against TNF-alpha-sensitive target cells. These findings demonstrate that TNF-alpha synthesis in macrophages is up-regulated by cGMP and down-regulated by cAMP, which indicates that cyclic nucleotides act as intracellular messengers for extracellular signals of macrophage activation.     

Effects of the medium ammonium-nitrate ratio on competence for asparagus cell division induced by phytosulfokine-α

The effects of medium ammonium-nitrate ratio on cell proliferation were investigated using a low cell-density culture of Asparagus officinalis L., which was triggered by a peptidal plant growth factor, phytosulfokine-α (PSK-α). The asparagus cells proliferated the most in a medium with an ammonium-nitrate ratio of 0:30 mM and could be maintained without significant loss of responsiveness to PSK-α for at least 96 h from the beginning of culture. In this medium, single cells gave rise to microcalli at initial densities as low as 3.2×10² cells/ml as long as PSK-α was present in the medium. Increasing the ammonium-nitrate ratio resulted in severe inhibition of cell proliferation at a low cell density, even if PSK-α was added to the medium. Received: 6 May 1997 / Revision received: 5 August 1997 / Accepted: 2 September 1997     

Staurosporine-induced cell death in Tetrahymena thermophila has mixed characteristics of both apoptotic and autophagic degeneration.

Staurosporine blocks signal transduction associated with cell survival, proliferation and chemosensory behaviour in the ciliated protozoan, Tetrahymena thermophila. Staurosporine inhibits cell proliferation and in vivo protein phosphorylation induced by phorbol ester. It also reduces the in vitro phosphorylation of the PKC-specific substrate, myelin basic protein fragment 4-14. Our results show that cell death in the presence of staurosporine is associated with morphological and ultrastructural changes similar to both apoptosis and autophagic degeneration, but these in turn can be postponed or prevented by 8-bromo-cyclic GMP, protoporphyrin IX, hemin or actinomycin D, although phorbol ester and insulin were ineffective. The results support the notion that staurosporine-induced cell death is an active process, associated with and/or requiring de novo RNA synthesis.     

Inhibition of cyclic GMP hydrolysis in human corpus cavernosum smooth muscle cells by vardenafil, a novel, selective phosphodiesterase type 5 inhibitor.

One of the key mediators of penile erectile function is nitric oxide (NO), which activates soluble guanylyl cyclase within the smooth muscle of erectile tissue and stimulates the production of cGMP. In addition to synthesis by cyclases, intracellular cGMP concentrations are tightly regulated by phosphodiesterases, which hydrolyze and inactivate cyclic nucleotides. In this study, we compared the inhibition of cGMP hydrolysis by vardenafil and sildenafil; two inhibitors selective for phosphodiesterase type 5 (PDE5). Vardenafil is a novel, high affinity PDE5 inhibitor currently under clinical development. In soluble extracts of human corpus cavernosum smooth muscle cells, vardenafil and sildenafil effectively inhibited cGMP hydrolysis at substrate concentrations of 1, 5 and 10 microM cGMP. The IC50 values for vardenafil were approximately 5-fold lower than for sildenafil at the substrate concentrations tested. Dixon plot analyses of the inhibition data demonstrated that vardenafil had a smaller inhibition constant (Ki = 4.5 nM) than sildenafil (Ki = 14.7 nM) in the same cellular extracts. In intact cells, 10 microM of the nitric oxide donor sodium nitroprusside resulted in a minimal (17%) increase in cGMP, relative to basal levels (321 +/- 65 fmol/mg prot). Treatment of cells with 10, 50 or 100 nM vardenafil, in the presence of 10 microM sodium nitroprusside, elevated cGMP levels in a dose dependent fashion, from 63% to 137% of basal levels. Equimolar concentrations of sildenafil also caused dose dependent increases in intracellular cGMP, but to a lesser extent (27-60%). These observations suggest that vardenafil is a more potent PDE5 inhibitor, than sildenafil in vitro. The more pronounced increase of cGMP in the presence of NO in intact cells suggests that vardenafil will be effective at lower doses than sildenafil under clinical conditions.     

Regulation of platelet cytosolic free calcium by cyclic nucleotides and protein kinase C

PAF elicits a rapid, concentration-dependent elevation of platelet cytosolic free calcium ([Caf]), measured by quin2. Elevation of [Caf] is transient, and the rate of reversal increases with agonist concentration. Adenylate cyclase stimulants (PGI2, PGD2) and 8-bromo cAMP; a guanylate cyclase stimulant (sodium nitroprusside) and 8-bromo cGMP; and a protein kinase C stimulant (phorbol myristate acetate) block the elevation of [Caf] induced by PAF, and accelerate its reversal. These results suggest that cAMP, cGMP and 1,2-diacylglycerol (DAG) could act as second messengers to regulate [Caf] in platelets. As PAF is known to stimulate platelet phosphoinositide hydrolysis (ergo DAG formation) but fails to elevate platelet cAMP or cGMP, it is proposed that DAG, via activation of protein kinase C, may act as an endogenous modulator of platelet [Caf]: an action that contributes to the role of DAG as a bi-directional regulator of platelet reactivity.     

Hemin-induced Erk1/2 activation and heme oxygenase-1 expression in human umbilical vein endothelial cells

Hemin has been reported to be protective in the pathological process, but its protective mechanisms have not been precisely defined. Hemin could induce Erk1/2 phosphorylation in astrocyte. Erk1/2 phosphorylation has been proved to be involved in many growth signals cellular transduction. However, little study has been conducted as to the relationship between hemin and Erk1/2 activation in human umbilical vein endothelial cells (HUVECs). The present study aimed to investigate the relationship between hemin and Erk1/2 phosphorylation in HUVECs. The results showed that low concentration of hemin induced and sustained phosphorylation of Erk1/2 for a long time. The HO inhibitor protoporphyrin IX zinc (II) abrogated phosphorylation of Erk1/2 induced by hemin. Biliverdin, one of the metabolites of hemin, obviously induced the Erk1/2 phosphorylation in HUVECs. Both hemin and biliverdin promoted HUVEC cell growth. The results strongly suggested that hemin could induce and sustain Erk1/2 phosphorylation in HUVECs by way of HO-1 induction and biliverdin produced from HO-1 catalysing hemin degradation.