Characterization of the binding of S-adenosyl-L-methionine to plasma membranes of HL-60 promyelocytic leukemia cells

S-Adenosyl-L-methionine (AdoMet) has been found to bind specifically to the plasma membrane of promyelocytic leukemia cells, HL-60. The Kd for AdoMet is 4.2.10(-6) M and the Bmax is 4.0.10(-12) mol/10(7) HL-60 cells. The binding is not related to the adenosine receptor since neither adenosine, ADP, nor ATP affect the ligand-receptor reaction. When HL-60 cells were incubated with physiological concentrations of [methyl-3H]AdoMet (20 microM) at 36 degrees C, AdoMet did not equilibrate with the intracellular pool, nor were any [3H]methyl groups incorporated into nucleic acids or proteins. In contrast, significant amounts of [3H]methyl groups were incorporated into membrane phospholipids. When cells were incubated with 20 microM [methyl-3H]AdoMet, [3H]methyl groups were transferred to phosphatidylethanolamine, -monomethylethanolamine, and -dimethylethanolamine yielding phosphatidylcholine. However, the rate of methyl transfer with AdoMet was only 22% of that observed when cells were incubated with a comparable amount of [methyl-3H]methionine. Both the binding of AdoMet and the methylation of phospholipids were inhibited by exogenous S-adenosyl-L-homocysteine. Therefore, the binding may be linked to a phospholipid methyltransferase.     

Identification of a tyrosine residue in rat guanidinoacetate methyltransferase that is photolabeled with S-adenosyl-L-methionine.

Exposure of rat guanidinoacetate methyltransferase to ultraviolet light in the presence of S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet) results in covalent linking of radioactivity to the enzyme protein. The incorporation of radioactivity shows no lag and is linear with respect to time up to 1 h. The photolabeling is saturable with [methyl-3H]AdoMet, and the binding constant of the enzyme for AdoMet determined in this experiment is similar to that obtained by equilibrium dialysis. Low concentrations of competitive inhibitors S-adenosyl-L-homocysteine and sinefungin effectively prevent the photoinduced labeling by AdoMet. Although guanidinoacetate methyltransferase is irreversibly inactivated upon ultraviolet irradiation in the absence of AdoMet, the enzyme inactivated by 1-h exposure to ultraviolet irradiation has been shown to bind AdoMet with an affinity identical to that of the native enzyme. These results indicate that photolabeling occurs at the active site. Following proteolysis of the [methyl-3H]-AdoMet-labeled enzyme with chymotrypsin, a radioactive peptide is isolated having a sequence Asp-Thr-X-Pro-Leu-Ser-Glu-Glu-Thr-Trp. The peptide corresponds to residues 134-143, with X being modified Tyr-136. The same peptide is photolabeled when [carboxy-14C]AdoMet is used. High-performance liquid chromatography of this peptide after acid hydrolysis and phenyl isothiocyanate derivatization suggests that the entire molecule of AdoMet is attached to Tyr-136.     

Incorporation of Intracisternally Administered l-[methyl-3H]Methionine and S-Adenosyl-l-[methyl-3H]-Methionine into Rat Brain Phospholipids

The incorporation of intracisternally injected L-[methyl-3H]methionine [( 3H]Met) or S-adenosyl-L-[methyl-3H]methionine (Ado[3H]Met) into rat brain AdoMet and phospholipid pools was examined. When [3H]Met was administered, both AdoMet and phospholipid pools were labeled. However, exogenously injected Ado[3H]Met did not serve as a substrate for phospholipid-N-methyltransferases. It was concluded that only Ado[3H]Met formed in situ was utilized to methylate phospholipids and that this process was initiated on the cytoplasmic side of the membrane. The apparent biological half-life in brainstem of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine formed from [3H]Met was 1.4 and 1.7 days, respectively. The half-life of phosphatidylcholine could not be determined due to interference from peripheral sources.     

Kinetics of S-adenosylmethionine cellular transport and protein methylation in Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

African trypanosomes of the Trypanosoma brucei group are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter for S-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates: Trypanosoma brucei brucei, a veterinary parasite, and Trypanosoma brucei rhodesiense, a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active against T. b. brucei. The apparent Km values for [methyl-3H]AdoMet transport, derived by Hanes-Woolf analysis, for T. b. brucei was 4.2 and 10 mM for T. b. rhodesiense, and the Vmax values were 124 and 400 micromol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/10(9) T. b. brucei and 3500 nmol/10(9) T. b. rhodesiense after 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation in T. b. brucei was fourfold greater than in T. b. rhodesiense. These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.     

Ubiquitin-Directed Antibodies Inhibit Neuronal Transporters in Rat Brain Synaptosomes

Affinity-purified antibodies specific for ubiquitin were found to inhibit the sodium-dependent uptake of [3H]choline, gamma-[3H]aminobutyric acid [( 3H]GABA), [3H]glutamate, [3H]norepinephrine, [3H]aspartate, and [3H]serotonin in rat cerebral cortical synaptosomes at a low concentration (10 micrograms/ml). These antibodies (termed anti-Ub) had no effect on the sodium-independent uptake of these substances or their calcium-dependent efflux. Synaptosomal [3H]deoxyglucose uptake was not affected in normal Krebs Ringer buffer containing 10 mM glucose, but was inhibited in glucose-free medium. Other nonneuronal sodium-dependent transport processes were found to be unaffected by 10 micrograms/ml anti-Ub, suggesting that anti-Ub does not bind indiscriminantly to sodium-binding sites on sodium-dependent organic solute transporters. Finally, anti-Ub inhibited sodium-dependent [3H]GABA and [3H]glutamate uptake in plasma membrane ghosts, devoid of membrane potential, which were derived from rat cerebral cortical synaptosomes. These results suggest that neuronal transporters or sites proximal to them may be ubiquitinylated on the plasma membrane surface.     

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.     

Further isolation of endogenous factors in canine and human plasma that inhibit [3H]norepinephrine accumulation

We have previously shown that both homologous canine plasma and a crude extract of this plasma contain substances that inhibit accumulation of [³H]norepinephrine ([³H]NE) by the canine saphenous vein. The purpose of this study was to further purify these substances and to determine if similar factors were present in human plasma. Crude extracts of plasma were purified with a Folch extraction in which most of the biological activity was recovered in the bottom or organic phase. This phase significantly inhibited [³H]NE uptake by the canine saphenous vein (23.5 ± 7.6% by concentrate from 9.1 ml of original plasma/ml incubate solution) and increased development of tension following transmural electrical stimulation by 91.5 ± 23.3% (extract from 1 ml of plasma/ml bath solution). The Folch extracts obtained from 100ml of plasma were purified by column and thin layer (TLC) chromatography. Samples were applied to a silicic acid column and eluted with chloroform, acetone, and methanol. The [³H]NE uptake inhibitory activity was primarily recovered in the methanol fraction. TLC of the methanol fraction of canine plasma on silica gel G plates (with pre-absorbent) resulted in five zones which were then assayed for their ability to inhibit [³H]NE accumulation by the saphenous vein. In the first zone (concentrate from 27.5 ml plasma/ml bath solution) there was significantly greater inhibitory activity (55.4 ± 8.3%), than in the corresponding zone obtained from solvent blanks (20.7 ± 4.1%). These results indicate that there is a factor or possibly factors in canine and human plasma that have thin layer chromatographic properties of a polar lipid, which inhibit [³H]NE accumulation and enhance the contractile response of vascular smooth muscle to transmural electrical stimulation.     

A radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-[methyl-3H] adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments of other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.     

Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase

We employed a highly specific photoaffinity labeling procedure, using (14)C-labeled S-adenosyl-l-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-(14)C]AdoMet or [carboxyl-(14)C]AdoMet yielded the sequence H(2)N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu(10654) and Pro(10655) as the major sites of derivatization. [carboxyl-(14)C]AdoMet in addition labeled Tyr(10650). Chymotryptic digestion generated the radiolabeled peptide H(2)N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu(2128) and Pro(2129), which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.     

Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.     

Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols

Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s-1, respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-l-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet. The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet. 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet. Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy. 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nm), suggesting that the successive C-methylation of the Delta 24 bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [2H3-methyl]AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on VCH3/VCD3 close to unity. [25-2H]24(28)-Methylenecycloartanol, [28E-2H]24 (28)-methylenelanosterol, and [28Z-2H]24(28)-methylene lanosterol were prepared and paired with AdoMet or [methyl-3H3]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Delta 24 bond.     

Enzymatic basis for the calcium-induced decrease of membrane protein methyl esterification in intact erythrocytes. Evidence for an impairment of S-adenosylmethionine synthesis

The effect of Ca2+ loading, induced by the ionophore A23187, on methyl esterification of membrane proteins (i.e. bands 2.1, 3, 4.1 and 4.5) has been investigated in intact human erythrocytes. When the cells were incubated with L-[methyl-3H]methionine, 40 microM CaCl2 and 10 microM A23187 induce a 50% inhibition of membrane protein methyl esterification. This effect is selectively due to the increased intracellular Ca2+ concentration, as it is antagonized by 10 mM EGTA, and other divalent cations such as Mn2+ do not exert any inhibition. In order to clarify the mechanism(s) of the reported inhibition, the various events involved in the methyl esterification process in vivo were analyzed. L-Methionine uptake as well as protein methylase II activity are not directly affected by altered intracellular Ca2+ concentrations. Conversely in the Ca2+-loaded erythrocytes the conversion of [3H]methionine into [3H]AdoMet, catalyzed by AdoMet synthetase, decreases up to 25%. When the undialyzed erythrocyte cytosolic fraction is assayed in vitro for AdoMet synthetase the activity of the enzyme from the CaCl2/A23187-treated erythrocytes is significantly lower than the control, up to 5 mM ATP. This result suggests that in the Ca2+-loaded erythrocytes the ATP intracellular concentration is significantly lowered. The direct evaluation of ATP intracellular concentration, by HPLC, confirms a significant drop of ATP level, as a consequence of the Ca2+ loading. The removal of Ca2+ from the cells quantitatively restores both the AdoMet synthesis and the methyl esterification levels. The possible role of altered ATP intracellular concentrations as a regulatory factor in the AdoMet-dependent reactions as well as in post-translational protein methylation related to the ageing process is also discussed.     

Purification to homogeneity of protein kinase C from bovine brain--identity with the phorbol ester receptor

The calcium- and phospholipid-dependent kinase activity (protein kinase C) was isolated from bovine brains by a combination of DEAE-cellulose chromatography, gel filtration and hydrophobic chromatography on octyl-Sepharose and phenyl-Sepharose. The phorbol ester receptor co-purifies with the protein kinase C throughout the procedure yielding a homogeneous protein of 79 500 daltons on SDS-polyacrylamide gels. The purified kinase incorporated approximately 5000 nmol phosphate into substrate/min/mg protein at saturating concentrations of Ca2+ and phosphatidyl serine. Reciprocal plots of protein kinase activity at varying phosphatidyl serine concentrations were biphasic and yielded two apparent Ka values for phosphatidyl serine of 0.6-2 and 35-80 micrograms/ml). These apparent Ka values were reduced 2- to 3-fold by either diolein (20 micrograms/ml) or phorbol-12,13-dibutyrate (10 micrograms/ml). The protein binds [3H]phorbol-12,13-dibutyrate ( [3H]PDB) with high affinity (Ka = 15 nM) in a phosphatidyl serine-dependent manner. At saturating phosphatidyl serine concentrations 0.89 mol [3H]PDB are bound per mol protein. The identification of protein kinase C as the phorbol ester receptor is discussed with respect to the function and regulation of this protein.     

Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester

Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Addition of [3H]CE-HDL to cells pretreated with adrenocorticotrophin in lipoprotein poor media resulted in a time- and concentration-dependent accumulation of [3H]cholesteryl ester and production of [3H]cholesterol and [3H]corticosterone. HDL-CE metabolism could be described as the sum of a high affinity ([ HDL-cholesterol]1/2 max = 16 micrograms/ml) and low affinity ([ HDL-cholesterol]1/2 max greater than 70 micrograms/ml) process. [3H]Cholesterol was found both intracellularly and in the media. Accumulation of [3H]cholesteryl ester could not be attributed to uptake and re-esterification of unesterified cholesterol since addition of Sandoz 58-035, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, did not prevent ester accumulation. Moreover, addition of chloroquine did not inhibit cholesteryl ester hydrolysis indicating that hydrolysis was not lysosomally mediated. Aminoglutethimide prevented conversion of [3H]CE-HDL to steroid hormones but did not inhibit [3H]cholesteryl ester uptake. Cellular accumulation of [3H] cholesteryl ester exceeded accumulation of 125I-apoproteins 5-fold at 1 h and 35-fold at 24 h indicating selective uptake of cholesteryl ester moiety. We conclude that rat adrenal cells possess a mechanism for selective uptake of HDL cholesteryl esters which provides substrate for steroidogenesis. These results constitute the first direct demonstration that cholesteryl esters in HDL can be used as steroidogenic substrate by the rat adrenal cortex.     

Ca2(+)-dependent synthesis of prostaglandin I2 and mobilization of arachidonic acid from phospholipids in cultured endothelial cells permeabilized with saponin

The feasibility of using saponin as a permeabilization agent to study the effect of free Ca2+ concentration ([Ca2+]f) on prostaglandin I2 (PGI2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 micrograms/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and beta-hexosaminidase. In cells prelabeled with [3H]arachidonic acid for 22 h, permeabilization with 20 micrograms/ml saponin induced PGI2 synthesis and release of [3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca2+]f in the range 72 nM to 5 microM. Release of [3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [3H]arachidonic acid and permeabilized with 20 micrograms/ml saponin. At [Ca2+]f optimal for PGI2 synthesis, 16.2% of the total incorporated [3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca2+]f of [3H]arachidonic acid release from phospholipids correlated with PGI2 synthesis. The amount of PGI2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI2 synthesis induced by A23187 was associated with less release of [3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca2+ dependence observed for PGI2 synthesis and [3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca2+ which culminate in PGI2 synthesis.     

Human Retinas Synthesize and Release Acetylcholine

Human retinas have the capacity to synthesize and release [3H]acetylcholine ([3H]ACh) after an incubation in [3H]choline ([3H]Ch). Synthesis of [3H]Ch by retinal homogenates was determined using either high-voltage paper electrophoresis (HVPE) or a two-step enzymatic/extraction assay for separating [3H]ACh from [3H]Ch. The enzymatic/extraction assay is shown to be accurate over a wide range of concentrations (10(-6)-10(-12) M). Homogenates of human retina synthesize [3H]ACh from [3H]Ch. We find an approximate Km of 50 microM and a Vmax of about 20 nmol/mg protein/h (at 37 degrees C) for the synthesis of labeled ACh by retinal homogenates. Human retinas also release [3H]ACh after a pulse of [3H]Ch. Release of labeled transmitter is stimulated by potassium depolarization. The potassium-stimulated release is partially blocked by magnesium or cobalt ions. Release data were analyzed by both the enzymatic/extraction assay and HVPE; the results are qualitatively identical in both cases. The data reported here provide additional evidence for cholinergic neurotransmission in the human retina.     

Characteristics of the action of prolactin on [3H]-thymidine incorporation into DNA in mammary gland explants from virgin mice

The prolactin stimulation of the rate of [3H]-thymidine incorporation into DNA in mammary gland explants from virgin C3H mice was studied. The onset of this effect occurred between one and two days after adding prolactin to the culture medium. Prolactin effected an enhanced rate of [3H]-thymidine incorporation at all concentrations from 10 ng/ml to 10 micrograms/ml. The response is essentially an "all or none" phenomenon since the effect at 10 ng/ml was not different from that at 10 micrograms/ml. Hydrocortisone was not essential from the prolactin response, but it did significantly increase the basal rate of [3H]-thymidine incorporation. Both quinacrine (an inhibitor of phospholipase A2 activity) and indomethacin (an inhibitor of prostaglandin biosynthesis) abolished the action of prolactin on [3H]-thymidine incorporation into DNA.     

Direct evidence that the insulin receptor mediates a mitogenic response in cultured human fibroblasts

To define the role of the insulin receptor in mediating a mitogenic response in cultured human fibroblasts, the effects of specific monoclonal antibodies against the insulin and the type I IGF receptor on insulin-stimulated [3H]thymidine incorporation were investigated. Insulin stimulated [3H]thymidine incorporation in a biphasic fashion. In the first phase, a half-maximal effect was observed at 20 ng/ml, and a seemingly maximal effect was obtained at 100-1000 ng/ml. With 10 micrograms/ml insulin, a secondary increase in [3H]thymidine incorporation was seen which was similar to the maximal effect of IGF-I. These [3H]thymidine incorporation results were corroborated with cell replication studies. MC-51, a highly specific monoclonal antibody for the insulin receptor, inhibited the stimulation of [3H]thymidine incorporation by 25 ng/ml of insulin. AlphaIR-3, a monoclonal antibody specifically directed against the type I IGF receptor, had no significant effect on insulin-stimulated [3H]thymidine incorporation at low (10-1000 ng/ml) concentrations of insulin. However, alpha IR-3 interfered with the incremental increase in [3H]thymidine incorporation observed at 10-100 micrograms/ml insulin. These data demonstrate that insulin, at low concentrations, is capable of stimulating DNA synthesis and replication of human fibroblasts through interaction with its own receptor, while at supraphysiological concentrations, much of insulin's mitogenic effect is mediated through the type I IGF receptor.     

Quantitative determination of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, in human plasma, using isotope dilution mass spectrometry

An original method is described for the determination in human plasma of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, by isotope dilution mass-spectrometry using 7,7-[2H2]-4-OHA as internal standard. This compound was synthesized starting from 7,7-[2H2]-4-androstene-3,17-dione. The procedure includes an extraction step using an Extrelut 1 column and a derivatization with N,o-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The minimum detection level of the method is 0.650 pg and the coefficients of variation for the 0.5 ng/ml (plasma) and 5 ng/ml (plasma) concentrations are 3.2% (within assay) and 6.7% (between assay) and 1.86% (within assay) and 2.3% (between assay) respectively.     

Platelet-activating factor mediates phosphatidylcholine hydrolysis by phospholipase D in human endometrium.

Human preimplantation embryos and endometrium secrete platelet-activating factor (PAF). The mechanism of phosphatidylcholine (PC) degradation stimulated by PAF was investigated in endometrial explants prelabeled with [methyl-3H]choline or preincubated with [3H]butan-1-ol. Analysis of the water-soluble metabolites of PAF-induced PC hydrolysis in secretory endometrium demonstrated that the stimulated generation of [3H]choline ([3H]Cho) precedes that of [3H]choline phosphate ([3H]ChoP) and [3H]glycerophosphocholine ([3H]GPC). Within 30 sec there was a rapid rise in PAF-induced [3H]Cho generation and by 2 min this had increased to 59.9% +/- 10.6% (p less than 0.02), with no effect upon [3H]ChoP and [3H]GPC during this period. Both [3H]GPC and [3H]ChoP, however, were increased at a later time point. The slower [3H]ChoP generation may suggest that PC-specific phospholipase C activation as well as delayed [3H]GPC rise may be due to PC-specific phospholipase A2 and lysophospholipase activation. Phospholipase D activity was confirmed by the incorporation of high-specific-activity [3H]butan-1-ol into [3H]phosphatidylbutanol ([3H]PBut). The rapid generation of [3H]PBut, which paralleled the rise in intracellular [3H]Cho, strongly suggests that PC breakdown is catalyzed by the phospholipase D pathway. It is proposed that PAF induces PC hydrolysis as a consequence of an early phospholipase D-catalyzed breakdown of PC in human secretory endometrium. This may be an alternative source for prostaglandin synthesis and an important pathway essential for long-term activation of local cellular events at the time of implantation.