Peroxynitrite formation from macrophage-derived nitric oxide.

Peroxynitrite formation by rat alveolar macrophages activated with phorbol 12-myristate 13-acetate was assayed by the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate. The inhibitor of nitric oxide synthesis N-methyl-L-arginine prevented the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate by stimulated macrophages, while Cu-depleted Zn superoxide dismutase did not catalyze the formation of 3-nitro-4-hydroxyphenylacetate either in vitro or in the presence of activated macrophages. The rate of phenolic nitration by activated macrophages was 9 +/- 2 pmol x 10(6) cells-1 x min-1 (mean +/- STD). Only 8% of synthetic peroxynitrite was trapped by superoxide dismutase, which suggested that the rate of peroxynitrite formation may have been as high as 0.11 nmol x 10(6) cells-1 x min-1. This upper estimate was consistent with N-methyl-L-arginine increasing the amount of superoxide detected with cytochrome c by 0.12 nmol x 10(6) cells-1 x min-1. The rate of nitrite and nitrate accumulation was 0.10 +/- 0.001 nmol x 10(6) cells-1 x min-1, suggesting that the majority of nitric oxide produced by activated macrophages may have been converted to peroxynitrite. The formation of a relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide in activated macrophages may contribute to inflammatory cell-mediated tissue injury.     

A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver

A sensitive method for the assay of sparteine oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km PM = 3033 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).     

Leukotriene B4 level in neutrophils from allergic and healthy subjects stimulated by low concentration of calcium ionophore A23187. Effect of exogenous arachidonic acid and possible endogenous source.

Peripheral blood neutrophils from patients with allergic rhinitis and from normal subjects were incubated for 5 min at 37 degrees C with 0.15 microM calcium ionophore A23187 in the absence or presence of exogenous arachidonic acid (2.5 to 10 microM). In neutrophils from allergic patients, the leukotriene B4 (LTB4) level was significantly increased by exogenous arachidonic acid in a concentration-dependent manner (16.2 +/- 4.2 and 38.1 +/- 6.8 pmol/5 min per 2 X 10(6) cells in the absence and presence of 10 microM arachidonic acid, respectively; P less than 0.005; n = 8). The LTB4 level in neutrophils from healthy subjects was only 0.97 +/- 0.17 pmol/5 min per 2 x 10(6) cells (n = 5) and was not enhanced by exogenous arachidonate. When cells from allergic patients were challenged in the presence of exogenous [1-14C]arachidonic acid, released LTB4 was radiolabeled and the incorporated radioactivity increased with the labeled arachidonate concentration. Labeled LTB4 was never detectable after incubating neutrophils from normal donors with exogenous labeled arachidonate. When neutrophils were incubated with [1-14C]arachidonate for 1 h, the different lipid pools of the two cell populations were labeled but both types of neutrophils produced unlabeled LTB4 in response to ionophore stimulation. The hydrolysis of choline and ethanolamine phospholipids into diacyl-, alkenylacyl- and alkylacyl-species revealed that solely the alkylacyl-subclass of phosphatidylcholine was unlabeled. We conclude (i) that neutrophils from allergic patients stimulated by low ionophore concentration produce more LTB4 than neutrophils from healthy subjects and incorporate exogenous arachidonate, (ii) that endogenous arachidonate converted to LTB4 by the 5-lipoxygenase pathway may provide only from 1-O-alkyl-2-arachidonoyl-glycero-3-phosphocholine.     

Comparison of the nuclear 5 alpha-reduction of testosterone and androstenedione in human prostatic carcinoma and benign prostatic hyperplasia.

The nuclear conversion of testosterone (T) to dihydrotestosterone (DHT) and androstenedione (delta 4A) to androstanedione (5 alpha-Adione) was compared in the separated stromal and epithelial fractions of hyperplastic (n = 6) and malignant (n = 3) prostatic tissues. Assay conditions were linear with respect to time and protein concentration and were optimal for NADPH concentration. The apparent Km values for the stromal enzymes were 0.2 and 0.02 microM for hyperplasia and carcinoma, respectively, using T as substrate. The apparent Km values, using delta 4A as substrate, were 0.03 and 0.02 microM, respectively. Apparent Vmax values for the stromal formation of DHT were 16.5 +/- 5.4 and 1.97 +/- 0.45 pmol/mg protein/30 min incubation, respectively, for the hyperplastic and malignant tissues. The apparent Vmax values for the formation of 5 alpha-Adione were 2.8 +/- 1.3 and 6.5 +/- 1.2 pmol/mg/protein/30 min incubation. The apparent Km values for the epithelial enzyme, for hyperplastic and malignant tissue were 0.04 and 0.04 microM, for T, and 0.05 and 0.03 microM for delta 4A. The respective apparent Vmax values were 4.6 +/- 0.93 and 0.65 +/- 0.07 for DHT and 2.0 +/- 0.86 and 6.4 +/- 0.45 pmol/mg protein/30 min incubation for 5 alpha-Adione. delta 4A was a competitive inhibitor of T 5 alpha-reduction. These results provide further evidence that different rates of 5 alpha-reduction at least partially explain the differences in androgen levels seen in the hyperplastic and the malignant prostate.     

Metabolism of arachidonic acid through the 5-lipoxygenase pathway in normal human peritoneal macrophages

Peritoneal macrophages (PM), obtained from 39 healthy women with normal laparoscopy findings, were stimulated with the ionophore A23187 or/and arachidonic acid (AA) both in adherence and in suspension. AA lipoxygenase metabolites were determined by reversed-phase HPLC. The major metabolites identified were 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene (LT)B4 and LTC4. The 20-hydroxy-LTB4, 20-carboxy-LTB4, and 15-HETE were not detected. Incubations of adherent PM with 2 microM A23187 induced the formation of LTB4, 110 +/- 19 pmol/10(6) cells, 5-HETE, 264 +/- 53 pmol/10(6) cells and LTC4, 192 +/- 37 pmol/10(6) cells. When incubated with 30 microM exogenous AA, adherent PM released similar amounts of 5-HETE (217 +/- 67 pmol/10(6) cells), but sevenfold less LTC4 (27 +/- 12 pmol/10(6) cells) (p less than 0.01). In these conditions LTB4 was not detectable. These results indicate that efficient LT synthesis in PM requires activation of the 5-lipoxygenase/LTA4 synthase, as demonstrated previously for blood phagocytes. When stimulated with ionophore, suspensions of Ficoll-Paque-purified PM produced the same lipoxygenase metabolites. The kinetics of accumulation of the 5-lipoxygenase/LTA4 synthase products in A23187-stimulated adherent cells varied for the various metabolites. LTB4 reached a plateau by 5 min, whereas LTC4 levels increased up to 60 min, the longest incubation time studied. Levels of 5-HETE were maximal at 5 min, and then slowly decreased with time. Thus, normal PM, in suspension or adherence, have the capacity to produce significant amounts of 5-HETE, LTB4, and LTC4. The profile of lipoxygenase products formed by the PM and the reactivity of this cell to AA and ionophore A23187 are similar to those of the human blood monocyte, but different from those of the human alveolar macrophage.     

Extracellular hydrolysis of diadenosine polyphosphates, ApnA, by bovine chromaffin cells in culture.

An ectoenzyme hydrolyzing diadenosine polyphosphates (ApnA) to AMP and Ap(n-1) has been studied in cultured chromaffin cells from bovine adrenal medulla. The KM value for extracellular Ap4A hydrolysis was 2.90 +/- 0.72 microM, the V(max) value obtained was 11.59 +/- 0.92 pmol/min x 10(6) cells (116 pmol/min.mg total protein). Ap3A, Ap5A, Ap6A, and Gp4G were competitive inhibitors of Ap4A hydrolysis with K(i) values of 3.65, 1.10, 1.20, and 2.65 microM, respectively. Phosphatidylinositol-specific phospholipase C removes the ApnA hydrolase activity from cultured chromaffin cells, suggesting an anchorage of this protein to the plasma membrane through the phosphatidylinositol. The turnover time for this enzyme calculated in the presence of cycloheximide was 38.94 +/- 1.53 hr for cultured chromaffin cells.     

Functional role of local angiotensin-converting enzyme (ACE) in adrenal catecholamine secretion in vivo

The aim of the present study was to investigate whether exogenous angiotensin I (AngI) is locally converted to angiotensin II (AngII), which in turn results in an increase in the adrenal catecholamine (CA) secretion in the adrenal gland in anesthetized dogs. Plasma CA concentrations in adrenal venous and aortic blood were determined by an HPLC-electrochemical method. Adrenal venous blood flow was measured by gravimetry. Local administration of AngI (0.0062 to 6.2 microg, 0.0096 to 9.6 microM) to the left adrenal gland resulted in significant increases in CA output in a dose-dependent manner. Following administration of 0.62 microg (0.96 microM) of AngI, adrenal epinephrine and norepinephrine outputs increased from 20.8+/-13.6 to 250.9+/-96.4 ng x min(-1) x g(-1) (p<0.05, n = 5) and from 2.8+/-1.7 to 29.6+/-11.1 ng x min(-1) x g(-1) (p<0.05, n = 5), respectively. From the same left adrenal gland, the output of AngII increased from -0.02+/-0.04 to 26.39+/-11.38 ng x min(-1) x g(-1) (p<0.05, n = 5), while plasma concentrations of AngII in aortic blood remained unchanged. In dogs receiving captopril (12.5 microg, 0.5 mM) 10 min prior to AngI, the net amounts of CA and AngII secreted during the first 3 min after AngI were diminished by about 80% (p<0.05, n = 5) compared with those obtained from the control group. There was a close correlation (r2 = 0.91, n = 6) between the net increases in AngII and CA outputs induced by AngI. The results indicate that the local angiotensin converting enzyme is functionally involved in regional AngII formation in the canine adrenal gland in vivo. The study suggests that AngII thus generated may play a role in the local regulation of adrenal CA secretion.     

Identification of an aldehyde dehydrogenase in the microsomes of human polymorphonuclear leukocytes that metabolizes 20-aldehyde leukotriene B4

We have previously reported that cytochrome P-450LTB in the microsomes of human polymorphonuclear leukocytes (PMN) catalyzes three omega-oxidations of leukotriene B4 (LTB4), leading to the sequential formation of 20-OH-LTB4, 20-CHO-LTB4, and 20-COOH-LTB4 (Soberman, R.J., Sutyak, J.P., Okita, R.T., Wendelborn, D.F., Roberts, L.J., II, and Austen, K. F. (1988) J. Biol. Chem. 263, 7996-8002). The identification of the novel final intermediate, 20-CHO-LTB4, allowed direct analysis of its metabolism by PMN microsomes in the presence of adenine nucleotide cofactors. Microsomes in the presence of 100 microM NAD+ or 100 microM NADP+ converted 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 with a Km of 2.4 +/- 0.8 microM (mean +/- S.E., n = 4) and a Vmax of 813.9 +/- 136.6 pmol.min-1.mg-1, for NAD+, as compared to 0.12 microM and 5.0 pmol.min-1.mg-1 (n = 2) for NADPH as a cofactor. The conversion of 1.0 microM of 20-CHO-LTB4 to 20-COOH-LTB4 in the presence of saturating concentrations (1.0 mM) of both NAD+ and NADP+ was not greater than the reaction in the presence of 1.0 mM of each cofactor separately, indicating that NAD+ and NADP+ were cofactors for the same enzyme. Antibody to cytochrome P-450 reductase did not inhibit the conversion of 20-CHO-LTB4 to 20-COOH-LTB4. When 1.0 microM 20-OH-LTB4 was added to microsomes in the presence of NADPH, approximately three-fourths of the product formed (63.7 +/- 5.1 pmol; mean +/- S.E., n = 3) was 20-CHO-LTB4 and approximately one-fourth (21.3 +/- 3.9 pmol; mean +/- S.E., n = 3) was 20-COOH-LTB4. In the presence of both NADPH and NAD+, only 20-COOH-LTB4 (85.5 +/- 9.9 pmol; mean +/- S.E., n = 3) was formed. PMN microsomes also contain an NADH-dependent aldehyde reductase which converts 20-CHO-LTB4 to 20-OH-LTB4, a member of the LTB4 family of molecules with biological activity. Based upon kinetic, cofactor and inhibition data, microsomal aldehyde dehydrogenase preferentially regulates the final and irreversible inactivation step in the LTB4 metabolic sequence.     

Comparison of nuclear 5 alpha-reductase activities in the stromal and epithelial fractions of human prostatic tissue

The nuclear conversion of testosterone (T) to dihydrotestosterone (DHT) was compared in the separated stromal and epithelial fractions of hyperplastic (n = 20), malignant (n = 5) and normal (n = 1) prostatic tissues. Standard assay conditions were: 1 microM testosterone, plus 4-6 X 10(5) DPM [3H]T, 1.0 mM NADPH, 2.0 mM EDTA and 0.5-1.0 mg nuclear protein in a total volume of 1.1 ml HEPES buffer, pH 7.4 (stroma) or MES buffer, pH 6.5 (epithelium). The apparent Km values for the stromal enzyme were 0.2, 0.2 and 0.3 microM, respectively, for the enzymes in hyperplastic, malignant and normal tissues. The Vmax values were 26 +/- 4.2, 2.8 +/- 0.6 and 4.1 pmol/mg protein/30 min incubation, respectively, for these same tissues. The apparent Km values for the epithelial enzymes, from the same tissues, were 0.03, 0.07 and 0.08 microM. The Vmax values for the epithelial enzymes were 4.8 +/- 1.2, 0.69 +/- 0.08 and 1.1 pmol/mg protein/30 min incubation. The pH optimum for the stromal enzyme lay between pH 6.5 and 7.5, whereas the pH optimum for the epithelial enzyme lay between 5.5 and 6.5. Enzymatic activity in both fractions revealed a biphasic response to zinc. In the absence of EDTA, microM quantities of zinc enhanced enzymatic activity while mM quantities inhibited this activity. These results would suggest that differences in the conversion of T to DHT help to explain, at least in part, the higher DHT levels seen in hyperplastic tissue and the higher T levels seen in the malignant prostate.     

Uptake and Metabolism of Serotonin by Ependymal Primary Cultures

Serotonin uptake and metabolism was studied in ependymal primary cultures. Serotonin uptake was facilitated by two different systems, one of which was the neuronal serotonin transporter SERT, exhibiting a Vmax value of 3.8+/-0.1 pmol x min(-1) x (mg protein)(-1) and an apparent Michaelis-Menten constant of 0.41+/-0.03 microM. The main product of metabolism was 5-hydroxyindole acetic acid, which resulted from the action of monoamine oxidase A. This enzyme showed a maximal rate of 0.85+/-0.02 nmol x min(-1) x (mg protein)(-1) and a Michaelis-Menten constant of 78+/-5 microM. Ependymal cells were able to dispose of extracellular serotonin with initial rates of approximately 600 pmol x min(-1) x (mg protein)(-1) and of 4 pmol x min(-1) x (mg protein)(-1) when challenged with 500 microM and 1 microM extracellular serotonin, respectively. Ependymal cells are concluded to facilitate the "sink" action of the CSF by removing waste compounds upon passing of the fluid from the parenchymal extracellular space into the ventricular system.     

Aromatase activity in human skin fibroblasts: characterization by an enzymatic method

Human skin fibroblasts were incubated for 24 h with 10(-6) M androstenedione and the estrone + estradiol released in the culture medium were measured by an enzymatic assay. Aromatase activity was expressed as pmol (estrone + estradiol) formed in the medium per mg cell protein per day. Using this method we were able to investigate the kinetic properties of aromatase in different cell strains and its stimulation by dexamethasone. Values of 92 nM and 9.1 pmol/mg protein/day were obtained respectively for Km and Vmax in cultured fibroblasts derived from genital skin of normal prepubertal subjects. In patients with complete androgen insensitivity syndrome CAIS, the Km was 156 nM and the Vmax 42 pmol/mg protein/day. Aromatase activity varied from 7.9 +/- 1.2 pmol/mg protein/day (mean +/- SD; n = 19) in normal prepubertal boys to 24.5 +/- 4.7 pmol/mg protein/day (mean +/- SD; n = 11) in those from normal postpubertal boys. The values were even higher in fibroblasts cultured from genital skin of prepubertal patients with CAIS. Cell concentrations did not modify the pattern of estrogen formation and aromatase activity did not vary with serial subcultures. The stimulatory effect of dexamethasone on aromatase activity in cultured fibroblasts was measured after preincubation of the cells for 48 h with dexamethasone, by determining estrogen formation after 24 h incubation of the cells with androstenedione 10(-6) M using this enzymatic method. This data suggest that aromatase activity measured in cultured fibroblasts could be a useful tool for studying extraglandular estrogen formation in physiological and pathological conditions.     

Vulgarenol, a sesquiterpene isolated from Magnolia grandiflora, induces nitric oxide synthases II and III overexpression in guinea pig hearts

Vulgarenol, a sesquiterpene isolated from Magnolia grandiflora flower petals, decreased coronary vascular resistance in the Langendorff isolated and perfused heart model, when compared to the control group [(15.2 x 10(7) +/- 1.0 x 10(7)) dyn s cm(-5) vs. (36.8 x 10(7) +/- 1.2 x 10(7)) dyn s cm(-5)]. Our data suggest that this coronary vasodilator effect probably involved inducible and endothelial nitric oxide synthase overexpression (6.8 and 4.2 times over control, respectively), which correlated with increases in nitric oxide release [(223 +/- 9) pmol mL(-1) vs. (61 +/- 11) pmol mL(-1)] and in cyclic guanosine monophosphate production [(142 +/- 8) pmol mg(-1) of tissue vs. (44 +/- 10) pmol mg(-1) of tissue], as compared to control values. This effect was antagonized by 3 microm gadolinium(III) chloride, 100 microM N-nitro-L-arginine methyl ester, and 10 microM 1H-[1,2,4]oxadiazolo[4,2-a]quinoxalin-1-one. Hence, the vulgarenol-elicited coronary vasodilator effect could be mediated by the nitric oxide-soluble guanylyl cyclase pathway.     

Adenosine transport in bovine chromaffin cells in culture

Bovine adrenal chromaffin cells in culture have a high capacity and affinity for adenosine uptake with Vmax = 14 +/- 2.4 pmol/10(6) cells/min (133 pmol/mg of protein/min) and Km = 1 +/- 0.2 microM. Transport studies, at short time periods, in recently isolated chromaffin cells have Vmax = 15 pmol/10(6) cells/min and Km = 1.1 microM in ATP-depleted cells. Endogenous levels of the various purine nucleosides and bases were determined by high pressure liquid chromatography, with adenosine (3 +/- 1 nmol/10(6) cells), inosine (5.3 +/- 1.2 nmol/10(6) cells), and hypoxanthine (2.1 +/- 0.8 nmol/10(6) cells) being the purine metabolites found in the highest concentration. Taking into account the intracellular water, endogenous levels of 2.1, 3.8, and 1.5 mM, respectively, were obtained. Radioactively labeled adenosine inside the cell underwent enzymatic transformations, producing inosine, hypoxanthine, xanthine, and nucleotides, with their appearance and distribution being a function of the incubation time. When nicotine was used as a secretagogue, the adenosine transformed into the nucleotide pool was released, reaching 18 +/- 8% of the total adenosine found in the nucleotides. Dipyridamole, extensively used clinically, was a strong inhibitor for the adenosine uptake into these cells, with Ki = 5 +/- 0.5 nM and noncompetitive kinetically.     

Platelet-activating factor induces leukotriene C4 synthesis by purified human eosinophils

Platelet-activating factor, at a concentration of 10 microM, was capable of inducing leukotriene C4 synthesis by eosinophils of healthy donors, i.e. (3.1 +/- 0.3) x 10(6) molecules leukotriene C4/cell (n = 31, mean +/- SEM, cell purity 87 +/- 2%). Reversed-phase high performance liquid chromatography analysis demonstrated the exclusive synthesis of leukotriene C4. At a concentration of 1 microM, platelet-activating factor was capable of significantly enhancing the calcium ionophore A23187, the opsonized zymosan or the arachidonic acid induced leukotriene C4 synthesis by eosinophils. These results show that PAF is capable of inducing and enhancing the leukotriene C4 formation by human eosinophils.     

Biochemical and morphological modifications in dexamethasone-treated mouse bone marrow-derived mast cells

Addition of 1 microM dexamethasone (DM) to bone marrow-derived mast cells (BMMC) induced a time-dependent increase in cell histamine content. The latter reached a plateau of 2.5 micrograms/1 x 10(6) cells after 11 days in culture, compared with 100 ng/1 x 10(6) for untreated BMMC. Steroids, such as beta-estradiol, androsterone, and testosterone (1 microM), did not alter the histamine content of BMMC, whereas progesterone (1 microM) induced a moderate increase. Other glucocorticosteroids also enhanced histamine content, suggesting that the observed increase was specific for glucocorticosteroid. Treatment of BMMC with 1 microM DM for 14 days inhibited the Ag-induced, IgE-mediated release of histamine, beta-hexosaminidase, platelet-activating factor-acether, LTB4, and LTC4 by 65 +/- 3%, 66 +/- 1%, 93 +/- 3%, 66 +/- 2%, and 74 +/- 10%, respectively (mean +/- 1 SD, n = 3). In contrast with untreated cells which produce less than 2 ng/1 x 10(6) cells PGD2 after Ag challenge, DM-treated BMMC generated 16.8 +/- 0.3 ng/1 x 10(6) cells PGD2. Moreover, most of DM-treated BMMC became Alcian blue+/safranin+ and by ultrastructure, exhibited numerous cytoplasmic granules filled with abundant and uniform electron-dense matrix. The present results indicate that DM-treated BMMC exhibit biochemical and functional properties different from immature untreated cells, suggesting that a maturation-like process occurred in vitro during DM treatment.     

Thienopyridines: effects on cultured endothelial cells.

In cultured endothelial cells harvested from human umbilical vein (HUVEC) or bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) caused an increase in nitrite generation in HUVEC from basal 227 +/- 37 to 372 +/- 60 or to 325 +/- 33 pmoles per 10(6) cells, respectively, and in BAEC from basal 182 +/- 17 to 378 +/- 18 or to 423 +/- 66 pmoles per 106 cells (n = 6), respectively. Calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) next to 30 min incubation with BAEC increased release of 6-keto-PGF 1alpha from basal level of 9.4 +/- 1.8 to 96.2 +/- 5.1 or to 99.5 +/- 10.2 pmoles per 10(6) cells, respectively. The pretreatment with aspirin (300 microM) cut down this rise to 4.2 +/- 0.1 pmoles per 10(6) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7 +/- 3.3 nM (n = 53), 68.3 +/- 5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cultured endothelial cells calcium ionophore A 23187 (0.1 microM) produced net maximum rise in [Ca2+]i by 157 +/-27 nM (n = 16)[ ECV304], by 107 +/- 58 nM (n=4) [HUVEC], and by 231.0 +/- 41.3 nM (n = 8) [BAEC], respectively, while ticlopidine (30 microM) produced net maximum rise in [Ca2+]i by 30.0 +/- 3.2 nM (n=9)[ECV304], 48.8 +/- 15.6 nM (n = 4)[HUVEC] and 28.4 +/- 5.4 nM (n = 8)[BAEC], respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than that of calcium A 23187 but also its maximum appeared after a lag period that was 2 3 times longer than that for A23187. In ECV304 clopidogrel at concentrations of 10, 30 and 100 microM produced maximum increment of [Ca2+]i by 16.5 +/- 3.8 nM (n = 7), 47.0 +/- 6.9 nM (n = 8) and 67.2 +/- 8.3 nM (n = 8), respectively. Incubation of BAEC with A23187 (microM), ticlopidine or clopidogrel (100 microM) for 2 h did not influence viability of cultured endothelial cells. We claim that thienopyridines, independently of their delayed anti-platelet properties ex vivo do release NO and PGI2 from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be mediated by a rise in [Ca2+]i, however, this possibility has not been proved.     

Physiological characterization of 45Ca2+ and 65Zn2+ transport by lobster hepatopancreatic endoplasmic reticulum

The crustacean hepatopancreas is an epithelial-lined, multifunctional organ that, among other activities, regulates the flow of calcium into and out of the animal's body throughout the life cycle. Transepithelial calcium flow across this epithelial cell layer occurs by the combination of calcium channels and cation exchangers at the apical pole of the cell and by an ATP-dependent, calcium ATPase in conjunction with a calcium channel and an Na+/Ca2+ antiporter in the basolateral cell region. The roles of intracellular organelles such as mitochondria, lysosomes, and endoplasmic reticulum (ER) in transepithelial calcium transport or in transient calcium sequestration are unclear, but may be involved in transferring cytosolic calcium from one cell pole to the other. The ER membrane has a complement of ATP-dependent calcium ATPases (SERCA) and calcium channels that regulate the uptake and possible transfer of calcium through this organelle during periods of intense calcium fluxes across the epithelium as a whole. This investigation characterized the mechanisms of calcium transport by lobster hepatopancreatic ER vesicles and the effects of drugs and heavy metals on them. Kinetic constants for 45Ca2+ influx under control conditions were K(n) (m)=10.38+/-1.01 microM, J(max)=14.75+/-1.27 pmol/mg protein x sec, and n=2.53+/-0.46. The Hill coefficient for 45Ca2+ influx under control conditions, approximating 2, suggests that approximately two calcium ions were transported for each transport cycle in the absence of ATP or the inhibitors. Addition of 1 mM ATP to the incubation medium significantly (P<0.01) elevated the rate of 45Ca2+ influx at all calcium activities used and retained the sigmoidal nature of the transport relationship. The kinetic constants for 45Ca2+ influx in the presence of 1 mM ATP were K(n) (m)=12.76+/-0.91 microM, J(max)=25.46+/-1.45 pmol/mg protein x sec, and n=1.95+/-0.15. Kinetic analyses of ER 65Zn2+ influx resulted in a sigmoidal relationship between transport rate and zinc activity under control conditions (K(n) (m)=38.63+/-0.52 microM, J(max)=19.35+/-0.17 pmol/mg protein x sec, n=1.81+/-0.03). The Addition of 1 mM ATP enhanced 65Zn2+ influx at each zinc activity, but maintained the overall sigmoidal nature of the kinetic relationship. The kinetic constants for zinc influx in the presence of 1 mM ATP were K(n) (m)=34.59+/-2.31 microM, J(max)=26.09+/-1.17 pmol/mg protein x sec, and n=1.96+/-0.17. Both sigmoidal and ATP-dependent calcium and zinc influxes by ER vesicles were reduced in the presence of thapsigargin and vanadate. This investigation found that lobster hepatopancreatic ER exhibited a thapsigargin- and vanadate-inhibited, SERCA-like, calcium ATPase. This transporter displayed cooperative calcium transport kinetics (Hill coefficient, n approximately 2.0) and was inhibited by the heavy metals zinc and copper, suggesting that the metals may reduce the binding and transport of calcium when they are present in the cytosol.     

Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ceramide stimulates epidermal growth factor receptor phosphorylation in A431 human epidermoid carcinoma cells. Evidence that ceramide may mediate sphingosine action

Recent studies suggest the existence of a signal transduction pathway involving sphingomyelin and derivatives (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies compare effects of ceramide, sphingosine, and N,N-dimethylsphingosine on epidermal growth factor (EGF) receptor phosphorylation in A431 human epidermoid carcinoma cells. To increase ceramide solubility, a ceramide containing octanoic acid at the second position (C8-cer) was synthesized. C8-cer induced time- and concentration-dependent EGF receptor phosphorylation. This event was detectable by 2 min and maximal by 10 min. As little as 0.1 microM C8-cer was effective, and 3 microM C8-cer induced maximal phosphorylation to 1.9-fold of control. EGF (20 nM) increased phosphorylation to 2.1-fold of control. Sphingosine stimulated receptor phosphorylation over the same concentration range (0.03-3 microM) and to the same extent (1.8-fold of control) as ceramide. The effects of C8-cer and sphingosine were similar by three separate criteria, phosphoamino acid analysis, anti-phosphotyrosine antibody immunoblotting, and phosphopeptide mapping by high performance liquid chromatography. Phosphorylation occurred specifically on threonine residues. N,N-Dimethylsphingosine, a potential derivative of sphingosine, was less effective. Since sphingosine and ceramide are interconvertible, the level of each compound was measured under conditions sufficient for EGF receptor phosphorylation. C8-cer (0.1-1 microM) induced dose-responsive elevation of cellular ceramide from 132 to 232 pmol.10(6) cells-1. In contrast, cellular sphingosine levels did not rise. This suggests that C8-cer acts without conversion to sphingosine. Exogenous sphingosine (0.1-1 microM) also increased cellular ceramide levels to 227 pmol.10(6) cells-1, but did not increase its own cellular level of 12 pmol.10(6) cells-1. Higher sphingosine concentrations that induced no further increase in EGF receptor phosphorylation produced very large elevations in cellular sphingosine. Hence, at effective concentrations, both compounds elevated cellular ceramide but not sphingosine levels. Additional studies performed with [3H]sphingosine demonstrated that cells contain substantially less N,N-dimethylsphingosine than free sphingosine and, during short term incubation, convert less than 5% of added sphingosine to N,N-dimethylsphingosine. These studies provide evidence that ceramide may have bioeffector properties and suggest sphingosine may act in part by conversion to ceramide.