Biomedical subjects

R A Coleman

Publications and source records attributed to R A Coleman.

At least 19 recordsLinked to original sources

PubMed2006

Relationship between bicarbonate retention and bone characteristics in broiler chickens.

Determination of the bicarbonate retention factor (BRF) is an important step during development of the indicator amino acid oxidation technique for use in a new model. A series of 4-h oxidation experiments were performed to determine the BRF of broilers aged 7, 14, 21, 28, 35, and 42 d using 4 birds per age group. A priming dose of 1.2 microCi of NaH(14)CO(3), followed by eight half-hourly doses of 1 microCi of NaH(14)CO(3) were given orally to each of 4 birds per age. The percentage of (14)C dose expired by the bird at a steady state was measured. These birds, as well as 12 additional birds matched for age and BW, were killed, and femur bone mineral density was measured by quantitative computed tomography to determine the relationship between bone development and bicarbonate retention at each age. There was a correlation (r = 0.50; P < 0.05) between total cross-sectional femur bone mineral density and bicarbonate retention at each age. A prediction equation (Y = 6.95 x 10(-2)X - 3.51 x 10(-5)X(2) + 27.58; P < 0.0001, R(2) = 0.79) where Y = bicarbonate retention and X = BW was generated to predict Y as a function of X. Bicarbonate retention values peaked at 28 d, during the stage of the most rapid bone deposition and the highest growth rate. A constant BRF was found from 1,900 to 2,700 g of BW of 35.15 +/- 1.095% (mean +/- SEM). This retention factor will allow the accurate correction of oxidation of (14)C-labeled substrates in broilers of different ages and BW in future indicator amino acid oxidation studies.

Aging↗

PubMed2005

Fibro-osseous pseudotumour of the digit--amputation for a benign but aggressive lesion.

Fibro-osseous pseudotumour is a rare, benign lesion which behaves aggressively and is, therefore, commonly misdiagnosed as a malignancy. Fibro-osseous pseudotumour shares many features with myositis ossificans and many consider it to be a subcutaneous variant of the latter. Early diagnosis and treatment by excision to normal tissue margins is important to preserve function and avoid digital amputation. We report a case of fibro-osseous pseudotumour of the finger which required ray amputation due to rapid progression of the lesion.

Aged↗

PubMed2002

5-HT(2B) receptors play a key role in mediating the excitatory effects of 5-HT in human colon in vitro.

1. 5-Hydroxytryptamine (5-HT) is known to produce a number of different effects in the gastrointestinal tract of various species, and has been proposed to play a key role in a number of intestinal disorders in man, including irritable bowel syndrome (IBS), although the receptors involved have yet to be established. The aim of the present study was to investigate the distribution and function of 5-HT(2B) receptors in human colon, and to establish their possible role in the aetiology of IBS. 2. The distribution of 5-HT(2B) receptor mRNA and protein were investigated by quantitative RT - PCR, Western analysis and immunocytochemistry. High levels of both mRNA and protein for 5-HT(2B) receptors were found throughout the human gastrointestinal tract, and in particular in colon, where 5-HT(2B) receptors were found predominantly in the longitudinal and circular smooth muscle layers within the muscularis externa, and in the myenteric nerve plexus lying between these two layers. 3. Electrical field stimulation of longitudinal muscle preparations of human colon mounted in organ baths resulted in neuronally-mediated contractile responses, that were significantly potentiated by application of 5-HT (up to 10(-7) M), with a pEC(50) of 8.2 +/- 0.1 (n=49 donors). The response to 5-HT was inhibited by a number of selective 5-HT(2B) receptor antagonists. 4. This study has shown for the first time that, in contrast to animal studies, the excitatory effects of 5-HT in human colon are mediated by 5-HT(2B) receptors. It is proposed that these receptors contribute to the putative 5-HT-induced colonic smooth muscle hypersensitivity associated with IBS.

Colon↗

PubMed2001

Regulation of mitochondrial sn-glycerol-3-phosphate acyltransferase activity: response to feeding status is unique in various rat tissues and is discordant with protein expression.

Triacylglycerol plays a critical role in an organism's ability to withstand fuel deprivation, and dysregulation of triacylglycerol synthesis is important in the development of diseases such as obesity and diabetes. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step of glycerolipid synthesis and is therefore a potential site for regulation of triacylglycerol synthesis. Because several studies suggest that triacylglycerol synthesis is linked to the mitochondrial isoform, we studied mitochondrial GPAT expression and the effect of feeding status on the regulation of mitochondrial GPAT in various rat tissues. Liver, adipose, and soleus muscle have high levels of GPAT mRNA, but low protein expression, whereas heart and adrenal, tissues with low GPAT mRNA abundance, have the highest GPAT protein expression. In addition, heart, which has the highest expression of mitochondrial GPAT protein, has low mitochondrial GPAT specific activity (0.02 nmol/min/mg). Liver and adipose have the highest mitochondrial GPAT specific activity (0.17 nmol/min/mg), but very low protein expression. Discrepancies between GPAT protein expression and activity suggest that mitochondrial GPAT may be regulated acutely. In response to a 48-h fast, liver and adipose mitochondrial GPAT protein expression and activity decrease 30-50%. After 24-h refeeding of either chow or high-sucrose diet, mitochondrial GPAT protein expression and activity overshoot normal levels 30-60%. In kidney, mitochondrial GPAT protein and activity increase 65 and 30%, respectively, with refeeding, whereas in the heart, mitochondrial GPAT activity increases 2.3-fold after a fast, with no change in protein expression. We also found that hepatic mitochondrial GPAT activity in the neonatal rat constitutes a lower percentage of the total GPAT activity than in the adult. We postulate that GPAT expression is modulated uniquely in each tissue according to specific needs for triacylglycerol storage.

Adipose Tissue↗

PubMed2001

Mitochondrial glycerol phosphate acyltransferase contains two transmembrane domains with the active site in the N-terminal domain facing the cytosol.

The topography of mitochondrial glycerol-3-phosphate acyltransferase (GPAT) was determined using rat liver mitochondria and mutagenized recombinant rat GPAT (828 aa (amino acids)) expressed in CHO cells. Hydrophobicity analysis of GPAT predicts two transmembrane domains (TMDs), residues 472-493 and 576-592. Residues 224-323 correspond to the active site of the enzyme, which is believed to lie on the cytosolic face of the outer mitochondrial membrane. Protease treatment of rat liver mitochondria revealed that GPAT has a membrane-protected segment of 14 kDa that could correspond to the mass of the two predicted TMDs plus a loop between aa 494 and 575. Recombinant GPAT constructs containing tagged epitopes were transiently expressed in Chinese hamster ovary cells and immunolocalized. Both the C and N termini epitope tags could be detected after selective permeabilization of only the plasma membrane, indicating that both termini face the cytosol. A 6-8-fold increase in GPAT-specific activity in the transfected cells confirmed correct protein folding and orientation. When the C terminus and loop-tagged GPAT construct was immunoassayed, the epitope at the C terminus could be detected when the plasma membrane was permeabilized, but loop-epitope accessibility required disruption of the outer mitochondrial membrane. Similar results were observed when GPAT was truncated before the second TMD, again consistent with an orientation in which the loop faces the mitochondrial intermembrane space. Although protease digestion of the HA-tagged loop resulted in preservation of a 14-kDa fragment, consistent with a membrane protected loop domain, neither the truncated nor loop-tagged enzymes conferred GPAT activity when overexpressed, suggesting that the loop plays a critical structural or regulatory role for GPAT function. Based on these data, we propose a GPAT topography model with two transmembrane domains in which both the N (aa 1-471) and C (aa 593-end) termini face the cytosol and a single loop (aa 494-575) faces the intermembrane space.

Animals↗

PubMed2001

Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty acids into triacylglycerol.

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [(14)C]oleate ( approximately 3 microm), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [(14)C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 microm oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.

Animals↗

PubMed2001

Expression and characterization of recombinant rat Acyl-CoA synthetases 1, 4, and 5. Selective inhibition by triacsin C and thiazolidinediones.

Inhibition by triacsins and troglitazone of long chain fatty acid incorporation into cellular lipids suggests the existence of inhibitor-sensitive and -resistant acyl-CoA synthetases (ACS, EC ) that are linked to specific metabolic pathways. In order to test this hypothesis, we cloned and purified rat ACS1, ACS4, and ACS5, the isoforms present in liver and fat cells, expressed the isoforms as ACS-Flag fusion proteins in Escherichia coli, and purified them by Flag affinity chromatography. The Flag epitope at the C terminus did not alter the kinetic properties of the enzyme. Purified ACS1-, 4-, and 5-Flag isoforms differed in their apparent K(m) values for ATP, thermolability, pH optima, requirement for Triton X-100, and sensitivity to N-ethylmaleimide and phenylglyoxal. The ACS inhibitor triacsin C strongly inhibited ACS1 and ACS4, but not ACS5. The thiazolidinedione (TZD) insulin-sensitizing drugs and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, troglitazone, rosiglitazone, and pioglitazone, strongly and specifically inhibited only ACS4, with an IC(50) of less than 1.5 microm. Troglitazone exhibited a mixed type inhibition of ACS4. alpha-Tocopherol, whose ring structure forms the non-TZD portion of troglitazone, did not inhibit ACS4, indicating that the thiazolidine-2,4-dione moiety is the critical component for inhibition. A non-TZD PPARgamma ligand, GW1929, which is 7-fold more potent than rosiglitazone, inhibited ACS1 and ACS4 poorly with an IC(50) of greater than 50 microm, more than 100-fold higher than was required for rosiglitazone, thereby demonstrating the specificity of TZD inhibition. Further, the PPARalpha ligands, clofibrate and GW4647, and various xenobiotic carboxylic acids known to be incorporated into complex lipids had no effect on ACS1, -4, or -5. These results, together with previous data showing that triacsin C and troglitazone strongly inhibit triacylglycerol synthesis compared with other metabolic pathways, suggest that ACS1 and ACS4 catalyze the synthesis of acyl-CoAs used for triacylglycerol synthesis and that lack of inhibition of a metabolic pathway by triacsin C does not prove lack of acyl-CoA involvement. The results further suggest the possibility that the insulin-sensitizing effects of the thiazolidinedione drugs might be achieved, in part, through direct interaction with ACS4 in a PPARgamma-independent manner.

3T3 Cells↗

PubMed2001

Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently.

Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC ) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5-2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations.

Amino Acid Sequence↗

PubMed2001

TH1 cytokine response of CD57+ T-cell subsets in healthy controls and patients with alcoholic liver disease.

Patients with chronic inflammatory diseases, including Crohn's disease and rheumatoid arthritis, as well as those with certain viral infections, and patients who are transplant recipients or who have certain hematologic malignancies have been observed to have CD57+ T cell expansion in both CD4+ and CD8+ subsets. We have reported previously that alcoholic patients also have CD57+ T cell expansion. Because many alcoholics become seriously deficient in cell-mediated immunity, it is of interest to determine whether the expanded CD57+ subsets can respond to stimulation with normal T helper cell subtype 1 (TH1) cytokine production. We report evaluation of the CD57 T-cell subsets of patients with alcoholic liver disease (ALD) with the use of cytoplasmic staining after stimulation through the T-cell receptor (TCR). The CD57+ subsets of the T cells of both healthy individuals and patients with ALD express significantly higher amounts of cytoplasmic tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) after 6 h of stimulation than do the CD57- subsets. This increased production can persist up to 46 h of continuous stimulation. Under these assay conditions, very little cytoplasmic interleukin (IL)-4 is observed in the T cells of either healthy control subjects or patients with ALD. Measurement of cytokine secretion by sort-purified CD57 T-cell subsets with the use of enzyme-linked immunosorbent assay (ELISA) shows that the CD57+ T-cell subset produces 18- to 30-fold more TNF- and IFN-, respectively, than does the CD57- subset in the first 12 h of stimulation. This response requires only stimulation through the TCR for the CD57+ subset, whereas significant secretion by the CD57- subset requires added IL-2 or anti-CD28 antibody. These results are consistent with the concept of the CD57+ T-cell subset as a differentiated effector cell and demonstrate that patients with ALD who are not drinking at the time of evaluation have normal or increased immediate TH1 T-cell responses.

Adult↗

PubMed2001

High glycemic index carbohydrate diet alters the diurnal rhythm of leptin but not insulin concentrations.

Morning serum leptin values in humans are inconsistently altered by diet, and the molecular mechanisms controlling the diurnal leptin pattern remain unexplained. We determined whether leptin values after meals or the leptin diurnal pattern was altered by the type of carbohydrate (CHO) ingested in diets containing either 20% or 30% fat. In a randomized, crossover study design, nine healthy lean adults ate one of four isocaloric diets for 8 days. Diets contained 15% protein: A, high glycemic index (GI) CHO, 30% fat; B, low GI CHO, 30% fat; C, high GI CHO, 20% fat; and D, low GI CHO, 20% fat. Serum glucose, insulin, and leptin were measured at intervals on Day 8 for 24 hr, and on Day 9 during an oral glucose tolerance test (GTT). Although the 24-hr glucose and insulin profiles did not differ with the diets, diets A and C altered the serum leptin diurnal pattern. In contrast to the usual evening rise in leptin concentration, which begins after 2200 hr, diets A and C caused a rise in leptin beginning at 1300 hr. The area under the curve for leptin between 1230 and 2400 hr was 17% greater for diets A and C. During the GTT, leptin concentrations were similar for each diet. These results suggest that the pattern and amount of leptin secretion may be altered by high GI CHO or the simple sugar content of the diet, unrelated to differences in insulin concentration, that high GI foods may have little or no effect on serum insulin in the context of a mixed meal, and that a single 0800-hr leptin value may not be sufficient to reveal a diet-induced change in leptin secretion

Adult↗

PubMed2001

Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis.

The diacylglycerol (DAG) signal generated from membrane phospholipids by hormone-activated phospholipases is attenuated by mechanisms that include lipolysis or phospholipid resynthesis. To determine whether the DAG signal might also be terminated by incorporation of DAG into triacylglycerol (TAG), we studied the direct formation of TAG from endogenous DAG generated by bacterial phospholipase C (PLC). When Chinese hamster ovary (CHO) cells prelabeled with [(14)C]oleate were treated with PLC from Clostridium perfringens for 6 h, [(14)C]phospholipid decreased 15% and labeled TAG increased 60%. This transfer of (14)C label was even greater when the cells were simultaneously exposed to PLC and 100 microM oleic acid. PLC as well as oleate treatment concomitantly increased the TAG mass within the cell. Moreover, when phospholipids were prelabeled with [(3)H]glycerol, a subsequent increase in [(3)H]TAG indicated that an intact DAG moiety was channeled into the TAG structure. Incubating CHO cells with the diacylglycerol kinase inhibitor R59022 enhanced the formation of TAG from phospholipids hydrolyzed by PLC or by PLC in the presence of 100 microM oleate, but not by incubation with oleate alone, indicating that the DAG released from plasma membrane phospholipids does not require the formation of a phosphatidic acid precursor for TAG synthesis. Similarly, the diacylglycerol lipase inhibitor RHC 80267 did not alter TAG synthesis from plasma membrane DAG, further supporting direct incorporation of DAG into TAG. These studies indicate that DAG derived from plasma membrane phospholipid is largely used for TAG formation, and support the view that this mechanism can terminate DAG signals. The studies also suggest that a transport mechanism exists to move plasma membrane-derived DAG to the endoplasmic reticulum.-Igal, R. A., J. M. Caviglia, I. N. T. de Gómez Dumm, and R. A. Coleman. Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis. J. Lipid Res. 2001. 42: 88;-95.

Acyltransferases↗

PubMed2000

Hoe 140 and pseudo-irreversible antagonism in the rat vas deferens in vitro.

The effects of bradykinin and the bradykinin B(2) receptor antagonists D-Arg-[Hyp(3),Thi(5,8),D-Phe(7)]-bradykinin (NPC 349) and D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-bradykinin (Hoe 140) were examined in the electrically-stimulated rat vas deferens. Cumulative additions of bradykinin (1-3000 nM) produced two distinct responses: an enhancement in the magnitude of the basal electrically-induced twitch response (neurogenic response) and an increase in the baseline tension (musculotropic response). NPC 349 (10-100 microM) produced concentration-dependent surmountable rightward shifts of both the bradykinin neurogenic and musculotropic response curves. In contrast, while Hoe 140 (10-100 nM) caused an apparently surmountable antagonism of the bradykinin neurogenic response, it caused an apparent insurmountable antagonism of the bradykinin musculotropic response. Interestingly, co-incubation of Hoe 140 (30 nM) with NPC 349 (30 and 100 microM) resulted in a concentration-related upwards displacement of the Hoe 140-suppressed bradykinin musculotropic response curve. Thus, Hoe 140 can be described as a pseudo-irreversible antagonist against the bradykinin musculotropic response. No time-dependent changes were observed in the maximum bradykinin musculotropic response attainable when NPC 349 (100 microM) additions were made for the final 2 or 18 min of the Hoe 140 incubation (20 min). These findings indicate that slow reversibility of Hoe 140 from the bradykinin B(2) receptor is unlikely to be the mechanism responsible for the pseudo-irreversible antagonism of the bradykinin-induced musculotropic response. Instead, we propose an alternative explanation involving a third, unstable and inactive form of the bradykinin B(2) receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2000

Palm tocotrienols protect ApoE +/- mice from diet-induced atheroma formation.

We evaluated the effects of vitamin E and beta-carotene on apolipoprotein (apo)E +/- female mice, which develop atherosclerosis only when fed diets high in triglyceride and cholesterol. Mice were fed a nonpurified control diet (5.3 g/100 g triglyceride, 0.2 g/100 g cholesterol), an atherogenic diet alone (15.8 g/100 g triglyceride, 1.25 g/100 g cholesterol, 0.5 g/100 g Na cholate) or the atherogenic diet supplemented with either 0.5 g/100 g (+)-alpha-tocopherol (mixed isomers); 0.5 g/100 g palm tocopherols (palm-E; 33% alpha-tocopherol, 16.1% alpha-tocotrienol, 2.3% beta-tocotrienol, 32.2% gamma-tocotrienol, 16.1% delta-tocotrienol); 1.5 g/100 g palm-E; or 0.01 g/100 g palm-carotenoids (58% beta-carotene, 33% alpha-carotene, 9% other carotenoids). Compared with mice fed the control diet, plasma cholesterol was fourfold greater in mice fed the atherogenic diet. Mice fed the 1.5 g/100 g palm-E supplement had 60% lower plasma cholesterol than groups fed the other atherogenic diets. Mice fed the atherogenic diet had markedly higher VLDL, intermediate density lipoprotein (IDL) and LDL cholesterol and markedly lower HDL cholesterol than the controls. Lipoprotein patterns in mice supplemented with alpha-tocopherol or palm carotenoids were similar to those of the mice fed the atherogenic diet alone, but the pattern in mice supplemented with 1. 5 g/100 g palm-E was similar to that of mice fed the control diet. In mice fed the atherogenic diet, the hepatic cholesterol plus cholesterol ester concentration was 4.4-fold greater than in mice fed the control diet. Supplementing with 1.5 g/100 g palm-E lowered hepatic cholesterol plus cholesterol ester concentration 66% compared with the atherogenic diet alone. Mice fed the atherogenic diet had large atherosclerotic lesions at the level of the aortic valve. With supplements of 0.5 g/100 g palm-E or 1.5 g/100 g palm-E, the size of the lesions was 92 or 98% smaller, respectively. The 0.5 g/100 g alpha-tocopherol and palm carotenoid supplements had no effect. Supplements did not alter mRNA abundance for apolipoproteins A1, E, and C3. The beneficial effect of tocotrienols on atherogenesis, the plasma lipoprotein profile and accumulation of hepatic cholesterol esters cannot be attributed to their antioxidant properties.

Animals↗

PubMed2000

Physiological and nutritional regulation of enzymes of triacylglycerol synthesis.

Although triacylglycerol stores play the critical role in an organism's ability to withstand fuel deprivation and are strongly associated with such disorders as diabetes, obesity, and atherosclerotic heart disease, information concerning the enzymes of triacylglycerol synthesis, their regulation by hormones, nutrients, and physiological conditions, their mechanisms of action, and the roles of specific isoforms has been limited by a lack of cloned cDNAs and purified proteins. Fortunately, molecular tools for several key enzymes in the synthetic pathway are becoming available. This review summarizes recent studies of these enzymes, their regulation under varying physiological conditions, their purported roles in synthesis of triacylglycerol and related glycerolipids, the possible functions of different isoenzymes, and the evidence for specialized cellular pools of triacylglycerol and glycerolipid intermediates.

Acyltransferases↗

PubMed2000

Acyl-CoAs are functionally channeled in liver: potential role of acyl-CoA synthetase.

Acyl-CoA synthetase (ACS) catalyzes the activation of long-chain fatty acids to acyl-CoAs, which can be metabolized to form CO(2), triacylglycerol (TAG), phospholipids (PL), and cholesteryl esters (CE). To determine whether inhibiting ACS affects these pathways differently, we incubated rat hepatocytes with [(14)C]oleate and the ACS inhibitor triacsin C. Triacsin inhibited TAG synthesis 70% in hepatocytes from fed rats and 40% in starved rats, but it had little effect on oleate incorporation into CE, PL, or beta-oxidation end products. Triacsin blocked [(3)H]glycerol incorporation into TAG and PL 33 and 25% more than it blocked [(14)C]oleate incorporation, suggesting greater inhibition of de novo TAG synthesis than reacylation. Triacsin did not affect oxidation of prelabeled intracellular lipid. ACS1 protein was abundant in liver microsomes but virtually undetectable in mitochondria. Refeeding increased microsomal ACS1 protein 89% but did not affect specific activity. Triacsin inhibited ACS specific activity in microsomes more from fed than from starved rats. These data suggest that ACS isozymes may be functionally linked to specific metabolic pathways and that ACS1 is not associated with beta-oxidation in liver.

Acyl Coenzyme A↗

PubMed2000

Expression of aquaporins in the renal connecting tubule.

The renal connecting tubule (CNT) is a distinct segment that occurs between the distal convoluted tubule (DCT) and the cortical collecting duct. On the basis of its characterization in rabbit it is widely believed that connecting tubule cells have a low permeability to water and do not respond to vasopressin. Here we utilize segment-specific markers and specific aquaporin antibodies to characterize expression of water channels in CNT of the rat by immunocytochemistry. Colocalization of aquaporin 2 (AQP2), AQP3, and AQP4 with Na(+), Ca(2+) exchanger (NCX), a transporter characteristic of the connecting tubule, gave heterogeneous labeling. There was aquaporin labeling in many but not all regions labeled by NCX. Colocalization of AQP2 with AQP3 and with AQP4 showed that AQP3 and AQP4 labeling were always accompanied by AQP2. Immunogold labeling and electron microscopy showed that NCX-labeled cells with AQP2 labeling had the morphology of CNT cells, whereas NCX-labeled cells without AQP2 labeling were DCT cells. The latter regions were identified as the late region of the DCT known as DCT2. Additionally, regions of CNT lacking AQP2 labeling could be identified in Brattleboro rats not treated with vasopressin but not in such animals chronically treated with deamino-Cys(1),D-Arg(8)-vasopressin (dDAVP). Quantitative analysis of labeling was consistent with expression of AQP2 over a longer region of CNT after dDAVP exposure.

Animals↗

PubMed2000

Measurement of cytochrome P450 gene induction in human hepatocytes using quantitative real-time reverse transcriptase-polymerase chain reaction.

Drug-induced changes in expression of cytochrome (CYP) P450 genes are a key cause of drug-drug interactions. Consequently, preclinical prediction of these changes by novel compounds is an integral component of drug development. To date, in vitro models of CYP induction have used mRNA measurement, immunodetection, and substrate metabolism as reporters. Here, we describe the application of quantitative real-time reverse transcriptase polymerase chain reaction to study CYP1A1 and CYP3A4 gene induction in 5-day-old cultures of human hepatocytes by known CYP inducers. After 5 days in culture, CYP1A1 expression was significantly elevated (5.1- to 26-fold; P <.01) in all four livers studied. In direct contrast, CYP3A4 mRNA levels consistently decreased during culture (80- to 300-fold; P <.001). In three independent experiments, a 48-h exposure to 3-methylcholanthrene, omeprazole, and lansoprazole significantly induced CYP1A1 expression in comparison to untreated cultures (P <.05). Rifampicin and solvent were without effect on CYP1A1 expression. Under identical experimental conditions, rifampicin and lansoprazole significantly elevated CYP3A4 mRNA expression (P <.05), whereas 3-methylcholanthrene, omeprazole, and dimethyl sulfoxide were without significant effect. These data demonstrate the applicability of quantitative reverse transcriptase polymerase chain reaction to the determination of gene dynamics in human hepatocytes. This offers a highly specific alternative to quantification of drug effects on CYP expression using immunodetection and substrate metabolism.

Base Sequence↗

PubMed2000

Characterization of muscarinic receptors in human lens cells by pharmacologic and molecular techniques.

PURPOSE: Activation of muscarinic receptors has been implicated in an increased risk of cataract after anticholinesterase treatment for glaucoma. The purpose of the present study was to determine the acetylcholine muscarinic receptor subtype(s) present in native human lens epithelial cells (NHLECs) and a human lens cell line, HLE-B3, and to compare the distribution in other ocular cells. METHODS: Human lens cells were perfused with artificial aqueous humor (35 degrees C) after fura-2 incorporation, and calcium levels were measured using a fluorometric single-cell digital imaging system. Acetylcholine was the primary muscarinic agonist, and the receptor subtypes were elucidated by determining the relative effectiveness of pirenzepine and AF-DX 384 in blocking the agonist-induced response. The levels of expression of mRNA for the receptor subtypes M1 through M5 were determined by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) using a sequence detection system (ABI Prism 7700; Perkin-Elmer, Foster City, CA). This was performed using total RNA extracted from native lens, retina, iris, and sclera and also cultured lens cells. RESULTS: Acetylcholine induced a similar concentration-dependent increase in peak-amplitude cytosolic calcium in the range 100 nM to 100 microM in both native and HLE-B3 cells. However, the kinetics of the response waveforms to 30-second pulses of acetylcholine were different in the two cell types. At higher concentrations (> 1 microM), a second phase appeared in the HLE-B3 cells that was absent in the NHLEC response. The 50% inhibitory concentration (IC50) values for blockade of a 1 microM acetylcholine response by pirenzepine and AF-DX 384 were 30 nM and 230 nM, respectively, for NHLECs, and 300 nM and 92 nM, respectively, for HLE-B3 cells. The QRT-PCR data showed that more than 90% of the total muscarinic receptor mRNA from NHLEC was of M1 origin. In the HLE-B3 cells, however, more than 95% of the mRNA was of M3 origin. mRNA for M3 was also in greatest abundance in other eye tissues, although there was a significant contribution from M1 in iris and sclera. CONCLUSIONS: Both NHLECs and HLE-B3 cells express muscarinic receptors that produce significant changes in cytosolic calcium in response to acetylcholine. Both pharmacologic and QRT-PCR evidence shows that whereas the M1 subtype predominates in NHLECs, M3 is the major contributor in HLE-B3 cells. In all other eye tissues, M3 appears to be the major contributor. These data should be taken into account when choosing particular models to investigate cataract mechanisms and also when designing muscarinic agonists to treat glaucoma.

Acetylcholine↗