In Vitro and In Vivo Metabolism and Pharmacokinetics of bis [(T-Butyl)-S-acyl-2-thioethyl]-β-L-2′,3′-dideoxy-5-fluorocytidine Monophosphate

Abstract

Exposure to 10 & M L-FddCMP-bisSATE led to formation of intracellular L-FddCTP levels of 410.1^± 46.2 and 242.1 ± 13.2 pmol/10⁶ cells in unstimulated and PHAstimulated PBM cells, respectively; whereas, exposure of cells to the parent nucleoside, L-FddC, generated 5-10-fold less L-FddCTP. In Hep-G2 cells and EGF/HGF stimulated and unstimulated primary cultured hepatocytes, the active metabolite reached 113 ± 29, 23.9 ± 15.6, and 20.6 ± 10.5 pmol/10⁶ cells. Three other metabolites, L-FddCMP-monoSATE, L-FddCMP-SH, and M I, were detected intracellularly and extracellularly in all cell types examined. Intravenous administered dose of 3 mg/kg L-FddCMP-bisSATE to rhesus monkeys resulted in plasma concentration levels of 2.06 ± 1.00 and 0.39 ± 0.15 & M of L-FddCMP-monoSATE and L-FddC, respectively, while the prodrug was completely cleared metabolically within 15 min. Following oral administration of an equivalent dose, the absolute oral bioavailability of L-FddC derived from L-FddCMP-bisSATE administration was 65%.     

Stimulation of phosphorylcholine turnover and diacylglycerol production in human polymorphonuclear leukocytes. Novel assay for phosphorylcholine.

Receptor-bypassing stimulants of human polymorphonuclear leukocytes (PMNLs), such as ionomycin or phorbol 12-myristate 13-acetate (PMA), generate an increase in diacylglycerol (DAG) which is independent of a phospholipase C specific for phosphatidylinositol 4,5,-bisphosphate (PIP2). Activation of a phospholipase C specific for phosphatidylcholine (PC) has been implicated as a source of DAG in other cells by measuring the release of radiolabelled phosphorylcholine. However, since PMNLs could not be labelled sufficiently with [3H]choline, we developed an h.p.l.c. assay to quantify mass levels of phosphorylcholine after enzymic conversion to [32P]CDP-choline with CTP-phosphorylcholine (choline phosphate) cytidylyltransferase (EC 2.7.7.15). This assay was linear to at least 20 nmol, and was sensitive to 10 pmol of phosphorylcholine. Baseline phosphorylcholine levels in unstimulated PMNLs were 2300 +/- 510 pmol/10(7) cells and were decreased by pretreatment with PMA (166 nM) or ionomycin (1 microM) for 10 min by 360 +/- 130 and 600 +/- 290 pmol/10(7) cells respectively (P less than 0.05). In contrast, baseline DAG levels were 147.6 +/- 11.7 pmol/10(7) cells in unstimulated PMNLs, and were increased by PMA or ionomycin by 1320 +/- 222 and 1891 +/- 264 pmol/10(7) cells respectively (P less than 0.05). Similarly, the chemoattractant fMet-Leu-Phe raised DAG levels by 731 +/- 111 pmol/10(7) cells and decreased phosphorylcholine levels by 180 +/- 60 pmol/10(7) cells. Activation of PMNLs by PMA, ionophore or fMet-Leu-Phe thus leads to the sustained production of DAG accompanied by the disappearance of phosphorylcholine. This suggests that these stimulants enhance PC turnover via a hydrolytic mechanism which is independent of phospholipase C, with activation of a PC-specific phospholipase D being a plausible mechanism.     

Receptors for D-Trp6-luteinizing hormone-releasing hormone, somatostatin, and insulin-like growth factor I in MXT mouse mammary carcinoma

Membrane receptors for D-Trp6-luteinizing hormone-releasing hormone (D-Trp6-LH-RH), somatostatin-14 (SS-14), and insulin-like growth factor I (IGF-I) were estimated in MXT mammary cancers of mice using sensitive multipoint micromethods. The receptors were characterized in untreated animals and following in vivo treatment with microcapsules of the agonist D-Trp6-LH-RH and the somatostatin analog RC-160, which strongly inhibited tumor growth. In the control group, D-Trp6-LH-RH was bound to the single class of saturable, specific, noncooperative receptor sites (Kd, = 29.3 +/- 8.48 x 10(-9) M; Bmax = 4.55 +/- 0.31 pmol/mg membrane protein). Treatment with D-Trp6-LH-RH alone or in combination with RC-160 produced down-regulation of membrane receptors for D-Trp6-LH-RH on MXT mammary tumor cells. RC-160 alone and ovariectomy were without effect on D-Trp6-LH-RH receptors. On the membrane surface of MXT mammary cells, we found one class of high affinity, specific, saturable binding sites for SS-14 (Kd = 4.4 +/- 1.9 x 10(-9) M; Bmax = 0.58 +/- 0.21 pmol/mg membrane protein). Treatment with RC-160 alone or combined with D-Trp6-LH-RH significantly increased both the dissociation binding constant (Kd = 18.6 +/- 3.5 x 10(-9) and 10.1 +/- 0.7 x 10(-9) M, respectively) and the binding capacity (Bmax = 13.98 +/- 1.7 and 21.00 +/- 4.0 pmol/mg membrane protein, respectively). We also found specific binding sites (Kd = 3.01 +/- 0.15 x 10(-9) M; Bmax = 2.24 +/- 0.96 pmol/mg membrane protein) for IGF-I in the membrane fractions of MXT mammary cancers. Chronic treatment with D-Trp6-LH-RH and RC-160 alone or in combination, as well as ovariectomy, significantly decreased the dissociation binding constant of IGF-I membrane receptors on MXT mammary cells. Our results strongly suggest an important role of LH-RH, SS-14, and IGF-I in the growth of MXT mammary carcinoma. Changes in characteristics of receptors after treatment with analogs of LH-RH and SS-14 along with tumor growth inhibition provide additional support for the direct effect of these peptides on tumor cells. A possible significance of these findings as applied to a clinical environment is discussed.     

Phospholipid hydroperoxide accumulation in liver of rats intoxicated with carbon tetrachloride and its inhibition by dietary alpha-tocopherol

The formation and accumulation of phospholipid hydroperoxides, especially of phosphatidylcholine hydroperoxide (PCOOH), a primary peroxidation product of phosphatidylcholine (PC), in livers of carbon tetrachloride-intoxicated rats was investigated. PCOOH in liver and blood plasma was measured by a chemiluminescence-high-performance liquid chromatography procedure originally developed by Miyazawa et al. (Anal. Lett. 20, 915, 1987; Free Radical Biol. Med. 7, 209, 1989). Male Sprague-Dawley rats (120 g body wt., 5 weeks of age) were used in the experiments. The amount of PCOOH in the liver of control rats (CCl4-untreated) was 160 +/- 20 pmol/100 mg protein (mean +/- SD) and the PCOOH/PC molar ratio was 1.1 +/- 0.1 X 10(-5). In CCl4 (0.1 ml/100 g body wt.)-dosed rats, the liver PCOOH was 289 +/- 65 pmol/100 mg protein (PCOOH/PC = 2.4 +/- 0.4 X 10(-5], 764 +/- 271 pmol/100 mg protein (PCOOH/PC = 5.2 +/- 1.7 X 10(-5], and 856 +/- 165 pmol/100 mg protien (PCOOH/PC = 6.0 +/- 0.8 X 10(-5] at 6 h, 24 h, and 1 week after the dose, respectively. Under such conditions, the liver phosphatidylethanolamine hydroperoxide (PEOOH) level was not altered and the concentration was less than 100 pmol/100 mg protein even after the dose. The increments of liver PCOOH were suppressed 56% by the oral supplementation of DL-alpha-tocopherol (5 mg/100 g body wt./day) for a week before CCl4 administration. A relatively larger amount of PEOOH was found after stimulation of PC hydroperoxidation in the liver of rats with a large amount of CCl4 (0.25 ml/100 g body wt.) rather than with the small amount of CCl4 (0.1 ml/100 g body wt.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of receptor-dependent activation of phosphatidylcholine hydrolysis by both phospholipase D and phospholipase C.

Enhancement of cellular phospholipase D (PLD)-1 and phospholipase C (PLC)-mediated hydrolysis of endogenous phosphatidylcholine (PC) during receptor-mediated cell activation has received increasing attention inasmuch as both enzymes can result in the formation of 1,2-diacylglycerol (DAG). The activities of PLD and PLC were examined in purified mast cells by quantitating the mass of the water-soluble hydrolysis products choline and phosphorylcholine, respectively. Using an assay based on choline kinase-mediated phosphorylation of choline that is capable of measuring choline and phosphorylcholine in the low picomole range, we quantitated the masses of both cell-associated and extracellular choline and phosphorylcholine. Activating mast cells by crosslinking its immunoglobulin E receptor (Fc epsilon-RI) resulted in an increase in cellular choline from 13.1 +/- 1.2 pmol/10(6) mast cells (mean +/- SE in unstimulated cells) to levels 5- to 10-fold higher, peaking 20 s after stimulation and rapidly returning toward baseline. The increase in cellular choline mass paralleled the increase in labeled phosphatidic acid accumulation detected in stimulated cells prelabeled with [3H]palmitic acid and preceded the increase in labeled DAG. Although intracellular phosphorylcholine levels were approximately 15-fold greater than choline in unstimulated cells (182 +/- 19 pmol/10(6) mast cells), stimulation resulted in a significant fall in phosphorylcholine levels shortly after stimulation. Pulse chase experiments demonstrated that the receptor-dependent increase in intracellular choline and the fall in phosphorylcholine were not due to hydrolysis of intracellular phosphorylcholine and suggested a receptor-dependent increase in PC resynthesis. When the extracellular medium was examined for the presence of water-soluble products of PC hydrolysis, receptor-dependent increases in the mass of both choline and phosphorylcholine were observed. Labeling studies demonstrated that these extracellular increases were not the result of leakage of these compounds from the cytosol. Taken together, these data lend support for a quantitatively greater role for receptor-mediated PC-PLD compared with PC-PLC during activation of mast cells.     

Gangliosides in the blood plasma: levels of ganglio-series gangliosides in the plasma after administration of brain gangliosides

The temporal change in the levels of the gangliotetraose-series gangliosides, i.e., GMla, GDla, GD1b, GT1b, in the blood plasma after intramuscular administration of bovine brain gangliosides (5 mg/kg) to beagle dogs (11.3-12.2 kg) was determined with high sensitivity by a recently developed thin-layer chromatography/enzyme-immunostaining method (Hirabayashi, Y., Koketsu, K., Higashi, H., Suzuki, Y., Matsumoto, M., Sugimoto, M. and Ogawa, T. (1986) Biochim. Biophys. Acta 876, 178-182). The amounts of GMla, GDla, GD1b, GT1b and their combined total in the plasma of beagle dogs before administration of gangliosides were 21 +/- 1, 36 +/- 7, 15 +/- 2, 16 +/- 2 and 88 +/- 6 pmol/ml of blood plasma, respectively. Trapezoidal calculation showed that the times of the maximum levels of GMla, GDla, GDlb, GTlb and the total of the their levels in the plasma were 8.0 +/- 1.2, 8.7 +/- 0.7, 6.3 +/- 2.0, 17.0 +/- 7.0 and 8.7 +/- 0.7 h after the administration of gangliosides, and their maximum concentrations were 517 +/- 37, 654 +/- 53, 160 +/- 5, 184 +/- 20 and 1383 +/- 74 pmol/ml, respectively. The maximum level of each ganglioside decreased gradually, reaching the normal level after 10 days. The half-maximum level of each ganglioside occurred 2-3 days after the administration. Asialo GM1 (GA1) was not detected plasma at any of the test times.     

Enhanced superoxide anion generation but reduced leukotriene B4 productivity in thioglycollate-elicited peritoneal macrophages

Lipoxygenase metabolism of arachidonic acid was compared between peritoneal macrophages from untreated rats and those from rats on day 7 after intraperitoneal injection of thioglycollate broth (TG). Resident macrophages (M phi) from untreated rats produced mainly LTB4 (303 +/- 25 pmol/5 x 10(6) cells) and 5-HETE (431 +/- 56 pmol/5 x 10(6) cells) when stimulated with 5 micrograms/ml calcium ionophore A23187 for 20 min at 37 degrees C. On the other hand, TG-elicited M phi generated less amounts of lipoxygenase metabolites (157 +/- 10 pmol LTB4 and 319 +/- 19 pmol 5-HETE/5 x 10(6) cells) with the same stimulus. Then, leukotriene productivity was examined by using subcellular fractions of each M phi lysate and an unstable epoxide intermediate, leukotriene A4. LTA4 hydrolase activity was mainly contained in soluble fractions from the both groups of M phi. The cytosol fraction from the resident M phi exhibited the following specific and total activity; 2.2 +/- 0.1 nmol LTB4/mg protein/5 min and 12.2 +/- 0.5 nmol LTB4/5 min per 10(8) cells. On the contrary, the cytosol fraction from the TG-elicited M phi showed 1.9 +/- 0.1 nmol LTB4/mg protein/5 min and 9.6 +/- 0.3 nmol LTB4/5 min per 10(8) cells. The resident M phi, however, generated 0.14 +/- 0.04 nmol O2-/min/4 x 10(5) cells whereas the TG-elicited M phi did 0.49 +/- 0.13 nmol O2-/min/4 x 10(5) cells when stimulated with wheat germ lectin. These results suggest that the TG-elicited macrophages show enhanced superoxide production but generate less lipoxygenase metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ornithine decarboxylase in resting human mononuclear leucocytes: evidence for an endogenous inhibitor

1. Ornithine decarboxylase (ODC) was measured in human mononuclear leucocytes (HML) by retention of putrescine on cation exchange paper. 2. The method was validated with unstimulated HML, phytohemagglutinin-stimulated HML, and a commercial preparation of ODC. The average enzyme activity of unstimulated HML (50 samples) was 22.6 +/- 7.3 pmol/hr 10(7) cells, with 29 values less than 5 pmol/hr 107 cells. 3. The results show that an endogenous inhibitor or inactivator of ODC exists in unstimulated HML: enzyme activity in extracts of mitogen-stimulated cells were inhibited by extracts of unstimulated cells (37-55%) inhibition under the conditions used.     

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.     

The aging Leydig cell: 1. Testosterone and adenosine 3',5'-monophosphate responses to gonadotropin stimulation in rats

Plasma testosterone levels before and after a single injection of hCG were significantly lower in 24-month old rats than 60--90 day old animals (p less than 0.001). Even with repeated hCG administration for three weeks, plasma testosterone levels of old rats could not be restored to levels present in unstimulated young rats. In response to in vitro LH and 8-bromo-cyclic AMP stimulation, purified young Leydig cells produced significantly higher amounts of testosterone than Leydig cells from old rats. Maximal testosterone formation of the young Leydig cells in response to LH was 42.0 +/- 6.88 ng/10(6) cells, while cells from old rats produced only 16.8 +/- 3.69 ng/10(6) cells (p less than 0.01). However, the dose of LH at which one half maximal response (ED50) occurred was 0.1 mIU/ml for young Leydig cells and 0.05 mIU/ml for old Leydig cells. Basal and 1.0 mIU LH-stimulated cyclic AMP formation were comparable in both groups, but cyclic AMP formation in response to 10 mIU of LH was significantly less in the old rats (p less than 0.05). Present results demonstrate impaired steroidogenic capacity of old rats both in vivo and in vitro. Decreased testosterone response in old rats most likely is the consequence of understimulation of Leydig cells by gonadotropin; however, there appear to be additional intrinsic defects in old Leydig cells.     

Pteridines are produced during interleukin 2-induced T-cell proliferation and modulate transmission of this signal

Pteridine levels of interleukin 2 (IL-2) receptor+ T-cell populations have been determined by HPLC after iodine oxidation; neopterin was monitored in the culture supernatants by radio-immunoassay. Upon addition of IL-2, cellular levels of biopterin and 6-hydroxymethylpterin rise transiently from 0.02 to 0.9 pmol/10(6) cells, cellular levels of neopterin from 1.5 to 4.1 pmol/10(6) cells. They peak at 8 and 13 h, respectively, after exposure to IL-2. Neopterin is not accumulated in the culture supernatant. DNA synthesis in T cells begins 10-12 h after adding the lymphokine and the portion of cells that undergo S-phase transition gradually increases during the subsequent 10 h. Entry into DNA synthesis phase is markedly accelerated if IL-2 is supplied together with tetrahydrobiopterin (0.8-1.6 X 10(-6) M) and the kinetics of entry into the S-phase transition during the period of 6-20 h become linear. This indicates that tetrahydrobiopterin modulation of IL-2 activity (Ziegler, I. et al. Naturwiss 72 (1985) 330) is an early event occurring during IL-2 signal transmission.     

Non-insulin dependent diabetic patients have increased glucose uptake in red blood cells

The kinetic characteristics of 3-O-methyl glucose (3-OMG) uptake were examined in red blood cells (RBC) from seven normal individuals (controls) and nine patients with non-insulin-dependent diabetes mellitus (NIDDM) treated with diet and oral hypoglycemic medication. Comparison of rates of 3-OMG uptake at 5 different substrate concentrations revealed significantly higher overall 3-OMG uptake in the diabetic group (P less than 0.0001). Kinetic parameters obtained for individual subjects showed there was not a significant difference in the Km between the diabetic (3.17 +/- 0.45 mM; mean +/- SE) and the control (2.46 +/- 0.25 mM) groups. However, Vmax was significantly increased (61%; P less than 0.025) in the diabetics (217.8 +/- 28.9 pmol/2 sec per 10(6) cells) compared to controls (135.2 +/- 15.6 pmol/2 sec per 10(6) cells). There was no correlation between HbA1C levels in the diabetic patients and Vmax values for 3-OMG uptake, suggesting that the increased hexose uptake was not accounted for simply by increased glycosylation in these cells. Glucose transport in RBC in hyperglycemic states may be a useful model for delineating the regulation of the non-insulin-mediated disposal of glucose in diabetes.     

Adenosine transporters in chromaffin cells: subcellular distribution and characterization

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.     

Liberation of cyclic AMP and catecholamine from the heart during left stellate stimulation in the anesthetized dog

In open-chest pentothal-chloralose anesthetized dogs, plasma catecholamine and cyclic AMP levels were evaluated in the aortic and coronary sinus blood, during stimulations of the left ansa subclavia (1, 2, and 4 Hz). Basal aortic and coronary sinus catecholamine levels were respectively 0.373 +/- 0.090 and 0.259 +/- 0.048 ng/mL and cyclic AMP levels averaged 21.4 +/- 1.4 and 20.9 +/- 1.6 pmol/mL. Statistically significant increases in cyclic AMP levels were induced by sympathetic stimulations at 1 Hz (2.0 +/- 0.6 pmol/mL, 2 Hz (2.5 +/- 1.2 pmol/mL) and 4 Hz (6.5 +/- 1.5 pmol/mL), concomitantly with elevations of coronary sinus catecholamine levels. Sotalol (5 mg/kg) abolished the increases in coronary sinus cyclic AMP levels induced in coronary sinus cyclic AMP output averaged 282 +/- 30 pmol/min (1 Hz), 662 +/- 160 pmol/min (2 Hz), and 1679 +/- 242 pmol/min (4 Hz). Sympathetically induced cyclic AMP output (4Hz) was blunted by sotalol (-81 +/- 14 pmol/min). Aortic cyclic AMP levels were not significantly influenced by stellate stimulation. Intense correlations were found between increased in coronary sinus plasma catecholamines and cyclic AMP concentration levels (r = 0.81, slope - 1.45, ordinate = -1.42, n = 15) as well as between delta cyclic AMP output versus delta catecholamine output values in the coronary sinus (r = 0.93. slope output levels. Intracoronary infusion of phenylephrine (10 micrograms/min) or nitroprusside (200 micrograms/min) had no influence on cyclic AMP plasma levels whereas aortic and coronary sinus levels were respectively increased 5.5 +/- 1.9 and 7.3 +/- 1.4 pmol/mL during the administration of isoproterenol (5 micrograms/min). These data suggested that plasma cyclic AMP constitutes a sensitive index of cardiac beta-adrenergic activity elicited by the release of endogenous catecholamine during stellate stimulations.     

Enhanced 5-lipoxygenase activity in lung macrophages compared to monocytes from normal subjects

We compared lipoxygenase activities of lung macrophages obtained from bronchoalveolar lavage to activities of blood monocytes purified by using discontinuous plasma/Percoll density gradients and adherence to tissue culture plastic in five normal subjects. Cells were incubated with ionophore A23187 (10(-9) to 10(-5) M) or arachidonic acid (0.12 to 80 microM) for 1 to 60 min at 37 degrees C to construct dose-response and time-dependence curves of lipoxygenase product generation. Products were identified and were quantified by using high-pressure liquid chromatography and ultraviolet spectroscopy. Under all conditions of product generation, both macrophages and monocytes generated predominantly (5S,12R)-dihydroxy-(6Z, 8E, 10E, 14Z)-eicosatetraenoic acid (leukotriene B4 (LTB4] and (5S)-hydroxy-(6E, 8Z, 11Z, 14Z) - eicosatetraenoic acid (5 - HETE), but, in each subject, macrophages invariably released greater amounts of LTB4 and 5-HETE than monocytes. In response to A23187, macrophages released a maximum of 183 +/- 96 pmol of LTB4 and 168 +/- 108 pmol of 5-HETE per 10(6) cells (mean +/- SEM), whereas monocytes released only 16 +/- 1 and 18 +/- 8 pmol per 10(6) cells of LTB4 and 5-HETE, respectively. After adding arachidonic acid, macrophages released a maximum of 52 +/- 21 pmol of LTB4 and 223 +/- 66 pmol of 5-HETE, whereas monocytes released no detectable products. The results suggest that mononuclear phagocyte maturation in the lung may be accompanied by an enhanced ability to generate 5-lipoxygenase products.     

The reduction in hepatic insulin clearance after oral glucose is not mediated by gastric inhibitory polypeptide (GIP)

Since the C-peptide/insulin ratio is reduced after oral glucose ingestion, the incretin hormone gastric inhibitory polypeptide (GIP) has been assumed to decrease hepatic insulin extraction. It was the aim of the present study to evaluate the effects of GIP on insulin extraction. Seventy-eight healthy subjects (27 male, 51 female, 43+/-11 years) were subjected to (a). an oral glucose tolerance test and (b). an intravenous injection of 20 pmol GIP/kg body weight, with capillary and venous blood samples collected over 30 min for insulin, C-peptide and GIP (specific immunoassays). Following GIP administration, plasma concentrations of total and intact GIP reached to peak levels of 80+/-7 and 54+/-5 pmol/l, respectively (p<0.0001). The rise in insulin after oral glucose and after intravenous GIP administration significantly exceeded the rise in C-peptide (p<0.0001). Estimating insulin extraction from the total integrated insulin and C-peptide concentrations (AUCs), only the oral glucose load (p<0.0001), but not the intravenous GIP administration (p=0.18) significantly reduced insulin clearance. Therefore, insulin clearance is reduced after an oral glucose load. This effect does not appear to be mediated by GIP.     

Stimulation of aromatase activity by dihydrotestosterone in human skin fibroblasts

In order to study the regulation of aromatase activity by androgens in cultured fibroblasts derived from genital skin of normal prepubertal boys, aromatase activity was evaluated in the presence of various concentrations of non-aromatizable androgen DHT(5 alpha-dihydrotestosterone). The estrogen formation was assayed by an enzymatic method, after 24 h incubation of the cells with 10(-6) M androstenedione. Aromatase activity was stimulated 3- to 20-fold by DHT at concentrations 10(-10) and 10(-9) M. It was necessary to preincubate the cells with DHT for 48 h in order to bring about this stimulation. The stimulatory effect was not significant after preincubation for only 24 h. The basal value of aromatase activity was in the range of 8 +/- 1.2 pmol/mg protein/day (mean +/- SEM), while the maximal stimulation 1043 +/- 46 pmol/mg protein/day was obtained at the concentration of 10(-8) M DHT. This stimulation was partially blocked with cyproterone acetate at level of 20 +/- 4 pmol/mg protein/day; stimulation of aromatase activity by DHT could thus be mediated by the androgen receptor. This stimulatory effect was prevented by incubation of the cells with cycloheximide or actinomycin D, suggesting that DHT acts to increase aromatase activity in cultured fibroblasts by inducing the synthesis of new proteinaceous material. In vitro regulation of aromatase activity by androgens could contribute to a new approach to the extraglandular formation of estrogen.     

3H-dopamine accumulation by rat brain synaptic vesicles in a membrane-impermeable medium

3H-Dopamine (DA) accumulation by storage vesicles from whole rat brain was significantly stablized in a buffer system based upon the membrane-impermeant D-potassium tartrate. 3H-DA uptake saturated by twenty minutes (Km 2.1 X 10(-5)M) and remained stable for periods of 40-60 minutes. Accumulated DA was rapidly exchangeable with exogenous DA. Total levels of accumulation (pmol/mg protein) were 41.7 +/- 2.9 (37 degrees), 11.9 +/- 2.5 (4 degrees), 31.3 +/- 1.8 (absence of ATP), 26.3 +/- 2.7 (reserpine, 10(-6)M), 26.1 +/- 0.67 (no ATP + reserpine 10(-6), and 14.6 +/- 2.4 (carbonylcyanide-p-triflouromethoxyphenylhydrazone, FCCP, 10(-6)M). Depletion of endogenous DA levels by pretreatment of the animals with alpha-methyl-p-tyrosine greatly diminished the reserpine-insensitive DA accumulation. After depletion of endogenous DA, ATP-independent uptake was significantly retarded, but eventually reached near-control levels. This uptake was abolished in the presence of FCCP (10(-6)M). The results suggest that endogenous levels of DA and ATP contribute to the reserpine- and ATP-insensitive DA accumulation observed in vesicles from untreated animals. HPLC analysis demonstrated no conversion of DA to norepinephrine (NE) in the course of the experiments.     

Characterization of the plasma membrane bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein coupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors

We have characterized the GTPase activity of the Ni-like guanine-nucleotide-binding regulatory protein in rabbit neutrophil plasma membranes. The low Km (3.64 +/- 0.87 X 10(-7) M) GTPase copurified with the formyl peptide receptor in the plasma membrane fraction obtained by discontinuous sucrose density gradient centrifugation. The Vmax (23.9 +/- 2.91 pmol/mg/min) and Km of the unstimulated enzyme were similar to those reported for Ni in other cell types. The activity of the unstimulated enzyme was both magnesium and sodium dependent and linear over the first 4 min of the assay. The chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and leukotriene B4 (LTB4) stimulated the GTPase in purified neutrophil plasma membrane preparations, whereas other secretagogues, such as A23187 and PMA, were without effect. Lineweaver-Burk analysis showed an fMLP-induced increase in Vmax (31.94 +/- 4.80 pmol/mg/min) (33.1 +/- 9.5%) but not in Km. The dose-response curve for fMLP stimulation showed an ED50 of 4.1 +/- 1.0 X 10(-8) M and an overall 22.2 +/- 3.1% maximal stimulation. C5a (30 micrograms/ml) increased the activity of the GTPase 21.3 +/- 5.7% and 10(-7) M LTB4 produced a 32.2 +/- 5.4% increase. Activated pertussis toxin treatment of neutrophil plasma membranes inhibited by 72.5 +/- 14.3% the stimulation of GTPase activity induced by fMLP; however, activated cholera toxin had no effect on the inhibition of fMLP stimulation, suggesting a direct role for an Ni-like protein in the coupling process. In contrast to the lack of inhibition of fMLP stimulation by activated cholera toxin treatment of plasma membranes, both pertussis toxin and to a lesser extent cholera toxin treatment reduced fMLP, C5a, and LTB4 stimulation of the GTPase in sonicates prepared from pretreated whole cells. Pertussis toxin inhibited fMLP stimulation of the GTPase by 75 +/- 7%, C5a stimulation was inhibited by 83 +/- 13%, and LTB4 stimulation was inhibited completely. Sonicates prepared from neutrophils treated similarly with cholera toxin showed a smaller inhibition of GTPase activity (50 +/- 4% and 14 +/- 9% for fMLP and LTB4, respectively) with the exception of C5a, where CT inhibition (81 +/- 32%) equaled pertussis toxin inhibition. Similarly, pertussis toxin completely inhibited the release of the granule enzyme N-acetyl-glucosaminidase by all three chemoattractants, whereas cholera toxin, except with C5a stimulation, had little or no effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells

A sensitive, nonradioactive analytical method has been developed to simultaneously determine the concentrations of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) in cultured cells. Following extraction, enzyme assays involving recombinant farnesyl protein transferase or geranylgeranyl protein transferase I are performed to conjugate FPP or GGPP to dansylated peptides. The reaction products are then separated and quantified by high-performance liquid chromatography coupled to a fluorescence detector at the excitation wavelength 335 nm and the emission wavelength 528 nm. The retention times for farnesyl-peptide and geranylgeranyl-peptide are 8.4 and 16.9 min, respectively. The lower limit of detection is 5 pg of FPP or GGPP ( approximately 0.01 pmol). A linear response has been established over a range of 5-1000 pg ( approximately 0.01-2 pmol) with good reproducibility. The method has been used to determine the levels of FPP (0.125+/-0.010 pmol/10(6)cells) and GGPP (0.145+/-0.008 pmol/10(6)cells) in NIH3T3 cells. Furthermore, changes in FPP and GGPP levels following treatment of cells with isoprenoid biosynthetic pathway inhibitors were measured. This method is suitable for the determination of the concentrations of FPP and GGPP in any cell type or tissue.