Purine metabolism in myeloid precursor cells during maturation. Studies with the HL-60 cell line.

In studies with the human promyelocytic leukemia cell line HL-60, we defined changes in intermediary purine metabolism that appear to contribute to the regulation of terminal maturation in myeloid cells. When HL-60 cells were exposed to compounds that induce maturation, consistent alterations in purine metabolism were found to occur within 24 h of culture. Perturbation of guanosine nucleotide synthesis and decreases of up to 50% in intracellular guanylate pool sizes were associated with the induced maturation of these cells in response to diverse inducing agents. While immature HL-60 cells were observed to synthesize purine nucleotides by both de novo and salvage pathways, the activity of both pathways decreased in cells induced to mature, although the relative contribution of purine salvage increased. Moreover, incorporation of the salvage pathway precursor, [14C]hypoxanthine from the intermediate, inosine monophosphate (IMP), into guanylates was reduced by approximately 65% in induced HL-60 cells, reflecting decreased activity of both hypoxanthine phosphoribosyltransferase and IMP dehydrogenase. When various inhibitors of IMP dehydrogenase (mycophenolic acid, 3-deazaguanosine, and 2-beta-D-ribofuranosylthiazole-4-carboxamide) were evaluated for their effects upon HL-60 cells, each agent was found to induce the cells to mature morphologically and functionally. Like other inducers, these agents decreased HL-60 cell proliferation and caused the cells to acquire an ability to phagocytose opsonized yeast and reduce nitroblue tetrazolium. Each agent reduced intracellular guanosine nucleotide pool sizes and induced HL-60 cell maturation at micromolar concentrations. These observations suggest that the size of intracellular guanosine nucleotide pools, the biosynthesis of guanosine nucleotides, and the activity of IMP dehydrogenase may be central to the regulation of terminal maturation in myeloid cells.

Survey of distribution of substance P, vasoactive intestinal polypeptide, cholecystokinin, neurotensin, Met-enkephalin, bombesin and PHI in the spinal cord of cat, dog, sloth and monkey.

Levels of substance P (sP), peptide-histidine-isoleucine (PHI), vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), neurotensin (NT), bombesin (BOM) and methionine-enkephalin (Met-Enk) like immunoreactivity were measured in cat, dog, primate and sloth cervical, thoracic, lumbar and sacral dorsal and ventral horns and dorsal root ganglia. The levels of peptides in the cat sacral cord and the principal peaks of immunoreactivity on a 10-60% acetonitrile gradient on a C18 reverse phase high performance liquid chromatography (HPLC) were sP (sP1-11: 369 ng/g), PHI (PHI: 271 ng/g), VIP (VIP1-28: 210 ng/g), Met-Enk (Met1-5 and extended forms: 257 ng/g), BOM (BOM1-10 and GRP1-27: 20 ng/g), CCK (CCK-8: 15 ng/g) and NT (NT1-13: 10 ng/g). Consideration of the rostrocaudal levels revealed an approximately even distribution with the exception of VIP and PHI which showed sacral/cervical ratios of 79 and 63. For sP, Met-Enk and BOM dorsal/ventral ratios were greater than 1 at all spinal levels. For VIP, PHI and CCK these ratios were greater than 1 only in the sacral cord. Dorsal root ganglion (DRG) levels of sP, VIP, PHI were readily measurable in single ganglia and covaried with the respective levels in the dorsal cord. Pooled samples of spinal ganglia and the trigeminal ganglia revealed that the relative levels of peptide immunoreactivity were: sP (25 ng/g); VIP (26 ng/g); PHI (28 ng/g); Met-Enk (6 ng/g); CCK (2 ng/g); NT (1 ng/g); and BOM (1 ng/g).

Cryptic Met-enkephalin in adrenal and portal vein during splanchnic artery occlusion shock in cats.

Adrenal vein (AD), portal vein (PV), and femoral artery (FA) plasma levels of immunoreactive (IR) Met-enkephalin pentapeptide (ME) and extended ME-IR forms, obtained after sequential incubation of plasma with trypsin and carboxypeptidase B, were examined in 4 cats during splanchnic artery occlusion shock at baseline (S1), during early shock (S2), late shock (S3), and after naloxone (1 mg/kg, i.v.) administration (S4). Early shock (S2) led to a significant increase in levels of extended and fully processed Met-enkephalin IR at all 3 collection sites (AD, PV, FA) without a change in proportional levels of extended Met-enkephalin IR to the pentapeptide IR (ME). Naloxone administration during late shock (S4), however, resulted in a disproportionate increase (150-fold from baseline) in adrenal vein plasma levels of extended Met-enkephalin IR forms, as compared to ME IR (23-fold). In contrast, no changes in plasma levels occurred in PV and FA.

Stimulated production of urokinase and plasminogen activator inhibitor-2 by the human promyelocytic leukemia cell line HL-60.

The effects of maturation inducing agents on the production of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) by the human promyelocytic leukemia cell line HL-60 were examined. PA activity, which was calibrated with a urokinase standard, was 3-6 mU/10(6) cells when measured in supernatants from control cells. This activity increased at least two-fold after dimethylformamide (DMF) or retinoic acid (RA) was added to cell cultures, and as much as ten to thirty-fold when cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (PMA), an agent that induces monocytoid differentiation in HL-60 cells. The PA activity produced by control and induced cells had the same molecular weight as urokinase (UK), and was completely inhibited by antibodies to UK. Cells that were induced with PMA but not with RA or DMF also produced an inhibitor to UK that was identified as PAI-2, the plasminogen activator inhibitor that is produced by monocytes. Because of its dual capacity to produce both UK and PAI, the HL-60 cell line represents a useful model for studies of the fibrinolytic mediators that are generated and released by leukemia cells.

Professor Robert F. Borkenstein - Appreciation of His Life and Work.

Few men have made such a significant and sustained contribution to their chosen field of endeavor as Robert F. Borkenstein. He is an international forensic science celebrity, highly respected by his peers. The two most well-known products of his brilliance are the Breathalyzer, invented in 1954, and the "Grand Rapids Study", a seminal work which established the relative risk of being involved in a traffic accident with various blood alcohol concentrations. In addition to his significant scientific achievements, he is greatly admired for his personal qualities, acute wit and total commitment to the cause of traffic safety. This article is a brief review and appreciation of his life and his many accomplishments.

Formation of the N'-methylnicotinamide adenine dinucleotide derivative of NAD in intact rat pituitary tumor GH3 and human promyelocytic leukemia HL-60 cells.

The NAD analog N'-methylnicotinamide adenine dinucleotide (N'AD) is formed in intact human promyelocytic leukemia HL-60 and in rat pituitary tumor GH3 cells during treatment of the cultured cells with the nicotinamide derivative N'-methylnicotinamide (N'CH3NAm). N'AD formation is associated with the induced maturation of HL-60 cells and increased hormone production by GH3 cells during treatment with the nicotinamide derivative. N'AD is detected by HPLC analysis of cytoplasmic extracts as a peak which elutes near NAD. Four facts indicate that this compound is N'AD. First, a compound which elutes with identical time retention is produced by transglycosylation during reaction of NAD with pig brain NAD glycohydrolase in the presence of excess N'CH3NAm. Second, the putative N'AD is degraded by prolonged digestion with the NAD glycohydrolase to ADP-ribose. Third, N'AD formation is prevented by addition of nicotinamide along with N'CH3NAm to compete with binding of N'CH3NAm to the NAD glycohydrolase. Fourth, radioactive precursor labeling demonstrates that it contains adenosine, but it is not labeled by radioactive nicotinamide. The biological relevance of N'AD formation was evaluated. The appearance of N'AD precedes development of HL-60 maturation, and NAD levels increase, not decrease, as observed in other cell types, during treatment with N'CH3NAm. Therefore, we propose that N'AD, not the pyridine base itself, is the active species in inducing maturation. The results provide support of a role for NAD metabolism, probably ADP-ribosylation, in the regulation of HL-60 maturation and in hormone production by pituitary cells.

Opioids preserve the adrenal medullary response evoked by severe hemorrhage: studies on adrenal catecholamine and met-enkephalin secretion in halothane anesthetized cats.

Possible modulatory effects of mu-, delta-, and kappa-receptor agonists on the concurrent adrenal secretion of catecholamines and met-enkephalin evoked by staged hemorrhage were examined in four groups of cats (n = 5 in each group) anesthetized with halothane (1 MAC). Group I received saline, group II received the mu-agonist sufentanil (25 micrograms/kg i.v., followed by a maintenance infusion), group III received the delta/mu agonist metkephamid (3 mg/kg i.v.), and group IV the kappa agonist U50488H (3.5 mg/kg i.v.). Samples for norepinephrine, epinephrine, dopamine, and met-enkephalin were taken simultaneously from the adrenal vein, femoral vein, and femoral artery at baseline, after drug administration, and after induction of 25% and 50% hemorrhage. In cats receiving saline, 25% hemorrhage resulted in a significant decline in mean arterial blood pressure (MABP) and no change in adrenal secretion. Fifty percent hemorrhage evoked no significant further fall in MABP, but led to prominent increases in adrenal vein hormone levels (norepinephrine, 30-fold; dopamine, 14-fold; and epinephrine, ten-fold) as compared to post-saline values. During the pre-hemorrhage baseline state, administration of sufentanil evoked a significant six- to 20-fold rise in adrenal vein catecholamine and met-enkephalin levels, whereas the administration of metkephamid and U50488H produced no change in adrenal secretion and a decrease in MABP. After 25% and 50% hemorrhage, there was no difference in adrenal vein hormone levels in cats receiving the mu-, delta-, or kappa-agonists compared to those receiving saline. No differences were observed in the different treatment groups with regard to the proportional levels of catecholamines and met-enkephalin in the adrenal vein during the course of the experiment. The authors conclude that opioids are not involved in the regulation of the secretory adrenal medullary response evoked by hemorrhage, and that the systems involved in mediating these cardiovascular reflexes differ pharmacologically from those systems mediating the autonomic response evoked by pain.

Maturation of human promyelocytic leukemia cells induced by nicotinamide: evidence of a regulatory role for ADP-ribosylation of chromosomal proteins.

We have studied the role of ADP-ribosylation of chromosomal proteins in the regulation of myeloid cell maturation using the HL-60 cell line as a model. Nuclei isolated from this human promyelocytic leukemia cell line contained (ADP-ribose)n synthetase activity, whereas little or no enzymatic activity was detectable in normal human blood neutrophils. Furthermore, the activity of (ADP-ribose)n synthetase was decreased in HL-60 cells when they were induced to mature with retinoic acid (RA). To determine whether reduced (ADP-ribose)n synthetase activity is simply a result of induced maturation or whether it is a necessary precedent event for the maturation process, we evaluated the effects of nicotinamide (NAm) and its methyl derivative, N'-methylnicotinamide (N'-Met-NAm), agents which decrease ADP-ribosylation. Treatment of HL-60 cells with these drugs caused the cells to undergo maturation and to acquire certain of the morphologic, functional, and biochemical characteristics of normal neutrophils. N'-Met-NAm was more potent than NAm in inducing maturation; at a concentration of 0.8 mM, it caused greater than 80% of the cells to mature, whereas a tenfold greater concentration of NAm was required to induce a similar degree of maturation. NAm and N'-Met-NAm also potentiated the maturation of HL-60 cells induced by RA. Exposure of cells to noninducing concentrations of these compounds caused a leftward shift in the dose-response curve for RA; maturation was observed at 10(-11) M RA in the presence of either 2 mM NAm or 0.2 mM N'-Met-NAm while 10(-9) M RA was required to induce maturation in their absence. A leftward shift in the dose response curve for maturation in the presence of low doses of NAm or N'-Met-NAm did not occur with another inducer, dimethyl formamide (DMF). Two enzymes, NAD glycohydrolase and tissue transglutaminase, that are abundant in macrophages, were induced by RA but not by NAm. N'-Met-NAm decreased by about 75% the amount of endogenous (ADP-ribose)n in a selected fraction of chromosomal proteins which included histone H1 and the nonhistone high mobility group proteins. The results of this study support the concept that ADP-ribosylation of chromosomal proteins influences the regulation of human myeloid cell maturation.

Induced maturation of the human promyelocytic leukemia cell line, HL-60, by 2-beta-D-ribofuranosylselenazole-4-carboxamide.

The new synthetic nucleoside analogue, 2-beta-D-ribofuranosylselenazole-4-carboxamide, was evaluated for its effects upon the growth and maturation of the human promyelocytic leukemia cell line, HL-60. At a concentration of greater than or equal to 1 nm, this agent was found both to decrease HL-60 cell proliferation and to cause the cells to acquire an ability to phagocytose opsonized yeast and to reduce nitroblue tetrazolium dye, functions characteristic of mature myeloid cells. In addition, this agent at similar concentrations caused a marked depression of intracellular guanosine nucleotide pools and a reduction in the incorporation of [14C] hypoxanthine into guanylates. These results suggested that the selenazole nucleoside caused an inhibition of inosinate monophosphate dehydrogenase, a key enzyme of guanylate biosynthesis. We therefore measured the activity of this enzyme indirectly by simultaneous-UV-radioactivity HPLC as well as by a direct radiometric method and demonstrated markedly reduced enzyme activities by both assays in drug treated cells. Dose response studies indicated that concentrations of drug which caused greater than 30% inhibition of IMP dehydrogenase activity induced greater than 50% maturation of the cells. These observations with this new nucleoside analogue provide further support for the concept that production of guanosine nucleotides and the activity of IMP dehydrogenase have a role in regulating the terminal maturation of myeloid cells.

Inosine monophosphate dehydrogenase and myeloid cell maturation.

In previous studies of purine ribonucleotide metabolism in the human myeloid leukemia cell line HL-60, we observed that there is a down-regulation of guanine ribonucleotide biosynthesis from the central intermediate, inosine monophosphate (IMP) and a depletion of intracellular guanosine triphosphate (GTP) and guanosine diphosphate (GDP) pools that occur during the induced maturation of these cells. We also found that inhibitors of IMP dehydrogenase, the enzyme that catalyzes the first step of guanylate synthesis from IMP, are potent inducers of HL-60 maturation. Because of these observations we specifically investigated the activity of IMP dehydrogenase in HL-60 cells and in a new inducible human myeloid leukemia cell line, RDFD2-25, both during maintenance culture and during induced maturation of the cells. Enzyme activity was examined directly in cell extracts with a radiometric assay that measures free 3H2O formed from [2-3H] IMP during the conversion of IMP to XMP. Uninduced HL-60 and RDFD2 cells in maintenance culture were found to have high levels of IMPD activity (5.2 to 5.7 pmol IMP metabolized/10(7) cells/min) compared with normal neutrophils and monocytes that had been purified from blood (less than 1.5 pmol IMP metabolized/10(7) cells/min). However, when HL-60 and RDFD2-25 cells were induced to mature with retinoic acid (10(-6) mol/L), dimethylformamide (6 X 10(-2) mol/L), or a known IMPD inhibitor, tiazofurin (10(-6) mol/L), IMPD activity in the cells fell by 51% to 80% within three to six hours. These changes in IMPD activity preceded detectable functional and antigenic maturation of the cells by at least 12 hours and were not temporally related to changes in cellular proliferation. These findings are consistent with the concept that the regulation of myeloid cell maturation may be influenced by intracellular concentrations of guanine ribonucleotides because IMP dehydrogenase activity is known to be rate limiting for the production of these nucleotides.

Effects of hemorrhage and naloxone on adrenal release of methionine-enkephalin and catecholamines in halothane anesthetized dogs.

Concurrent levels of methionine-enkephalin and catecholamines in adrenal vein, femoral vein and femoral artery were measured under baseline conditions and during graded hemorrhage in halothane anesthetized dogs and compared to a non-bled control group. Naloxone was administered in both groups at the end of the experiment. Normotensive hypovolemia with a remaining blood volume of 76% led to a moderate decrease in mean arterial blood pressure from baseline and a 15- to 20-fold increase in norepinephrine, epinephrine and dopamine, and a 5-fold increase in enkephalin in the adrenal vein. Subsequent induction of hypotensive hypovolemia with a remaining blood volume of 51% resulted in a profound drop in blood pressure and evoked a further increase in the level of catecholamines (40- to 50-fold from baseline) and enkephalin (8-fold from baseline) in the adrenal vein. In the control group only a 3- to 4-fold increase from baseline in adrenal vein hormone levels was observed over time. Naloxone administration at the end of the experiment, led to a 2- to 6-fold further increase in hormones at the 3 collection sites in both groups of dogs. Joint calculation of the partial correlation coefficients for the influence of preceding blood volume and blood pressure, and concurrent blood volume and blood pressure on hormone secretion in the adrenal vein revealed that these variables explained the variation in hormone levels between 56 and 92% during normotensive hypovolemia and 62-83% during hypotensive hypovolemia. In one dog with bilateral adrenalectomy, hemorrhage was poorly tolerated, and naloxone administration did not lead to increased systemic plasma levels of catecholamines and enkephalin or improved hemodynamics. In the hemorrhage group, molar ratios of norepinephrine/epinephrine in the adrenal vein showed a significant increasing trend during the experiment. Findings in these experiments support the idea of differential monoaminergic and enkephalinergic regulation in adrenal medullary cells.

Neural isolation of the jejunoileum. Effect on tissue morphometry, mucosal disaccharidase activity, and tissue peptide content.

The aim of this study was to determine the effects of a model of intestinal extrinsic denervation on mucosal structure and function. Six dogs underwent in situ neural isolation of the jejunoileum (Group 2); six other dogs served as operated controls (Group 1), and five nonoperated dogs were naive controls (Group 3). Thirty-centimeter segments of proximal jejunum and distal ileum were excised before (time zero) and at 2 weeks and 8 weeks postoperatively in Groups 1 and 2, while similar regions were removed at time zero in Group 3. Tissues were analyzed for morphology with quantitative morphometry, mucosal disaccharidase activities (sucrase, maltase, and lactase), and tissue content of selected regulatory peptides in transmural, mucosa/submucosa, and muscularis regions. In situ neural isolation had no significant or consistent effects on morphology/morphometry or on mucosal disaccharidase activities. Tissue content of neuropeptide Y decreased markedly (P < 0.002) in all layers of the jejunal and ileal walls, but tissue content of vasoactive inhibitory polypeptide, substance P, cholecystokinin, neurotensin, met-enkephalin, neurokinin A, somatostatin, and calcitonin gene-related peptide demonstrated only minor changes. The physiologic effects of intestinal transplantation (extrinsic denervation and disruption of intrinsic, enteric neural continuity, and lymphatic drainage) have little effect on morphology, mucosal disaccharidase activity, and tissue content of most regulatory peptides. How these minor alterations might affect enteric function, however, needs to be investigated.

The effects of electroconvulsive therapy on melatonin.

Although several studies have investigated the impact of various antidepressant medications on melatonin, there are no published reports addressing the effects of electroconvulsive therapy (ECT). Melatonin's major urinary metabolite, 6-sulfatoxymelatonin (6MT), was measured before and after an acute course of ECT. Fourteen subjects diagnosed with major depression who had failed prior pharmacologic therapy were enrolled. 6MT excretion was measured using an enzyme-linked immunosorbent assay test in 24 hour samples separated into daytime and nighttime components. Hamilton Rating Scale for Depression scores showed a significant improvement (p < 0.0001). Data analysis using the Wilcoxon signed rank test demonstrated a significant decrease in 24 hour 6MT post-ECT (p < 0.016) and daytime 6MT (p < 0.008). These results demonstrate an association between a therapeutic response to ECT and decrease in endogenous melatonin production.

Distribution and correlates of plasma fibrinogen in middle-aged women. Initial findings of the Postmenopausal Estrogen/Progestin Interventions (PEPI) study.

Fibrinogen levels have been reported in cohort and case-control studies to be positively related to the development of coronary heart disease. This report presents the distribution and determinants of fibrinogen in women enrolling in a 3-year randomized trial of hormone replacement therapy (HRT), the Postmenopausal Estrogen/Progestin Interventions (PEPI) trial. Fasting plasma fibrinogen levels were measured in 874 postmenopausal women, aged 45 to 65 years, who had not used HRT for at least 3.5 months. Mean (+/- SD) fibrinogen level was 2.83 +/- 0.45 g/L. There was a significant positive association between fibrinogen and age (P = .03). Significantly higher (P < .005) fibrinogen levels were seen in current smokers versus nonsmokers (2.94 versus 2.81 g/L), in women who reported consuming fewer than 12 alcoholic drinks in the 12 months before the baseline visit versus higher consumption (2.90 versus 2.79 g/L), and in women who reported never versus ever having used HRT (2.90 versus 2.77 g/L). Self-reported leisure time physical activity (LTPA) was negatively associated (P = .0001) with fibrinogen levels as follows: inactive (2.84 g/L), light (2.89 g/L), moderate (2.80 g/L), and heavy (2.60 g/L), with significantly (P = .0001) lower levels in women who reported heavy LTPA versus each of the other categories and in women reporting moderate versus light LTPA. A strong positive correlation was found between fibrinogen and body mass index (BMI) (r = .32; P < .0001). In a model that included age, smoking, alcohol intake, prior HRT, LTPA, and BMI, LTPA was no longer a statistically significant predictor of fibrinogen level, while associations with other variables remained significant.(ABSTRACT TRUNCATED AT 250 WORDS)