Cyclic AMP-stimulated fluid transport in the thyroid: influence of thyroid stimulators, amiloride and acetazolamide on the dynamics of domes in monolayer cultures of porcine thyroid cells

Confluent monolayer cultures of porcine thyroid cells form dome-shaped elevations by local separation from the plastic culture dish. Formation of domes by epithelial cells in culture is generally considered to be evidence of fluid transport. A computer-controlled data acquisition system was developed to quantitate fluid transport in thyroid cultures by serial measurements of dome elevation. Thyrotrophin (10 mU/ml), prostaglandin E2 (PGE2; 0.01-1 mumol/l), forskolin (1 mumol/l), 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (0.5 mmol/l) and 3-isobutyl-1-methyl-xanthine (0.5 mmol/l) promoted increases in dome height over 5-120 min. Dome growth in the presence of PGE2 (1 mumol/l) was inhibited by amiloride (0.1-100 mumol/l), ouabain (200 mumol/l), or by removal of bicarbonate and glucose from the medium. In media of reduced bicarbonate concentration (1 mmol/l compared with the control concentration of 10 mmol/l), dome growth was inhibited by acetazolamide (0.01-1 mmol/l). These data are consistent with cyclic AMP-stimulated transport of fluid from apical to basal pole of the cells, dependent on sodium entry through the apical pole by an Na+/H+ exchanger.     

Bile acid transport by basal membrane vesicles of human term placental trophoblast

The aim of this work was to investigate the first step in the vectorial translocation of bile acids from the fetus to the mother, which is the transfer across the basal (i.e., fetal-facing) plasma membrane of the trophoblast. Thus, the uptake of [14C]taurocholate by basal plasma membrane vesicles obtained from normal human term placentas was studied. Taurocholate retention into vesicles was studied using a rapid filtration technique that was modified to reduce the taurocholate binding to the filters and to the external surface of the vesicles. Using 100 mumol/L substrate, the membrane vesicles showed a temperature-dependent, Na(+)-independent transport of taurocholate into an osmotically reactive intravesicular space. The initial rate of taurocholate influx in the presence of 100 mmol/L KNO3 followed saturation kinetics (apparent Km for taurocholate = 670 +/- 128 mumol/L; Vmax = 1.86 +/- 0.28 nmol/mg protein.60 s at 37 degrees C). Over the 6.9-7.9 pH range neither internal nor external pH nor inward nor outward proton gradients affected the uptake of taurocholate. When the electrical potential difference across the basal membrane was manipulated by external anion replacement (Cl-, SCN-, SO4(2-), or NO3-) or by valinomycin-induced K(+)-diffusion potential (vesicle inside negative), taurocholate uptake was not significantly modified. Taurocholate uptake was cis-inhibited in the presence of 1 mmol/L glycocholate, 0.5 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonate and 0.5 mmol/L sulfobromophthalein. However, 1 mmol/L probenecid or 0.5 mmol/L p-aminohippurate had no effect. Moreover, preloading the vesicles with 100 mmol/L HCO3- (but not with 100 mmol/L Cl- or 50 mmol/L SO4(2-) induced a significant enhancement in the initial rate of taurocholate uptake. In summary, these findings provide strong evidence for the presence of an electroneutral transport system for taurocholate in the basal plasma membrane of human chorionic trophoblast. They also suggest that this is likely to be an anion-exchange system.     

Guanylin strongly stimulates rat duodenal HCO3- secretion: Proposed mechanism and comparison with other secretagogues

BACKGROUND & AIMS: Guanylin and heat-stable enterotoxin (STa) stimulate intestinal Cl- secretion via activation of the cystic fibrosis transmembrane regulator (CFTR)-encoded Cl- channel. It was speculated that CFTR activation also regulates electrogenic duodenal HCO3- secretion. Therefore, the effect of guanylin/STa and other secretagogues on rat duodenal HCO3- secretion was studied. METHODS: The HCO3- secretory rate of in vitro rat proximal duodenum was determined by pH stat titration and paracellular permeability by 3H-mannitol fluxes, bidirectional 36Cl- fluxes were measured, and the short-circuit current (Isc) was recorded. RESULTS: Luminal guanylin and STa concentration dependently stimulated the HCO3- secretory rate and Isc. Guanylin-stimulated HCO3- secretion was independent of luminal Cl-, inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)- benzoate, and additive to the HCO3- secretory rate stimulated by glucagon and carbachol but not by the tested adenosine 3',5'-cyclic monophosphate (cAMP)-dependent agonists. The ratio of the HCO3- secretory rate/Isc stimulated by the tested guanosine 3',5'-cyclic monophosphate (cGMP)-dependent agonists was markedly higher than the cAMP-dependent agonists. Prostaglandin E2 and 8-bromo-cAMP but not STa/guanylin also transiently increased paracellular permeability. CONCLUSIONS: Guanylin and STa stimulate electrogenic HCO3- secretion in rat duodenum, most likely via CFTR Cl- channel activation, but the different relationship for HCO3- to Isc in cGMP-than in cAMP-stimulated anion secretion suggests a different cellular source and/or signaling pathways. (Gastroenterology 1996 Dec;111(6):1558-68)     

Endotoxin-induced alterations in rat colonic water and electrolyte transport.

This study examines the effects of endotoxin on intestinal water and electrolyte transport in adult male rats. Endotoxin (1.55 mg/kg, intravenously) reduced in vivo colonic saline absorption 61% in 1 hour. In vitro unidirectional and net 22Na and 36Cl fluxes showed that endotoxin significantly decreased net colonic 22Na absorption compared with control colons (0.3 +/- 1.7 vs. 4.8 +/- 1.1 microEq/h x cm2). Although endotoxin had no significant effect on basal short circuit current (Isc) and conductance, 3H-inulin flux studies suggested an increase in colonic permeability. Isc responses to the 5'-cyclic adenosine monophosphate (cAMP)-dependent secretagogues prostaglandin E2 (1 mumol/L) and vasoactive intestinal peptide (0.1 mumol/L) were diminished by 80% and 50%, respectively. However, cytosolic cAMP-dependent protein kinase activity under basal and stimulated (6 mumol/L 8-bromo-cAMP) conditions was not altered by endotoxin treatment. The Isc responses to 10 mumol/L bethanechol, a Ca(2+)-dependent agonist, were not effected by endotoxin treatment. It was concluded that endotoxin significantly affects colonic transport function and may contribute to the development of diarrhea in inflammatory bowel diseases.     

Papaverine inhibits transcytotic vesicle transport and lipid excretion into bile in isolated perfused rat liver.

Papaverine is a nonspecific smooth muscle relaxant and a phosphodiesterase inhibitor. Its effects on biliary excretion of lipids and horseradish peroxidase were investigated in a single-pass isolated perfused rat liver model. A constant infusion of papaverine (1.6 mumol/min; 40 mumol/L) significantly increased bile flow (microliters per minute per gram of liver) before (2.03 +/- 0.09 vs. 1.0 +/- 0.06) and after sodium taurocholate infusion (2.77 +/- 0.10 vs. 1.88 +/- 0.11). However, papaverine significantly and reversibly reduced biliary excretion of phospholipids and cholesterol (nanomoles per minute per gram of liver) after a 1.0 mumol/min sodium taurocholate infusion, from 7.45 +/- 0.83 and 1.42 +/- 0.15 to 1.75 +/- 0.18 and 0.39 +/- 0.06, respectively (p less than 0.01), whereas secretion of bile acids was unaffected. When a 1-min pulse of horseradish peroxidase (25 mg) was infused in isolated perfused rat liver after a continuous infusion of N6,O-2'-dibutyryladenosine 3',5'-cyclic monophosphate (0.25 mumol/min; 6.25 mumol/L), horseradish peroxidase appeared in bile in an early (4 to 6 min) and late (20 to 25 min) peak. Papaverine significantly reduced the late peak, from 1.211 +/- 0.264 to 0.498 +/- 0.107 (p less than 0.01). Papaverine had no significant effects on either cyclic AMP or cyclic GMP in the liver and bile, although it has been reported that papaverine is a phosphodiesterase inhibitor. These findings indicate that papaverine inhibits biliary excretion of lipids but not bile acids, and they suggest that papaverine has an inhibitory effect on transcytotic vesicle transport independent of an increase of cyclic nucleotides in hepatocytes.     

Impairment of noninsulin-mediated glucose disposal in the elderly

While normal aging is characterized by resistance to insulin-mediated glucose disposal (IMGU), the effect of age on noninsulin-mediated glucose disposal (NIMGU), which is responsible for the majority of basal glucose uptake, has not been completely evaluated. These studies were conducted on healthy nonobese young (n = 10; age, 20-30 yr) and old (n = 10; age, 62-80 yr) men. Each subject underwent two paired studies in random order. In all studies a [3H]glucose infusion was used to measure glucose uptake and production rates, and somatostatin (500 micrograms/h) was infused to suppress endogenous insulin release. In study A, plasma glucose was kept close to fasting levels (approximately 5.6 mmol/L) using an euglycemic clamp protocol for 4 h. Plasma insulin decreased to less than 20 pmol/L within 15 min and remained suppressed thereafter in all studies. Steady state (15-240 min) plasma glucagon levels were slightly greater in the elderly [young, 86 +/- 5 (+/- SE); old, 98 +/- 2 ng/L; P less than .05]. Basal glucose uptake was similar in both groups (young, 877 +/- 21; old, 901 +/- 24 mumol/min). Glucose uptake during the last hour of the study (180-240 min) was used to represent NIMGU, because insulin action was assumed to be absent by this time. NIMGU was less in the elderly (young, 744 +/- 18; old, 632 +/- 32 mumol/min; P less than 0.01). In study B, plasma glucose was kept at about 11 mmol/L for 4 h using a hyperglycemic clamp protocol. Plasma insulin decreased to less than 20 pmol/L within 15 min and remained suppressed thereafter in all studies. Steady state plasma glucagon levels were slightly but not significantly higher in the elderly (young, 88 +/- 6; old, 100 +/- 4 ng/L). Basal glucose uptake (young, 910 +/- 27; old, 883 +/- 25 mumol/min) and NIMGU (young, 933 +/- 36; old, 890 +/- 16 mumol/min; P = NS) were similar in both young and old subjects. We conclude that aging is associated with impairment in NIMGU only in the basal state, which may explain in part the increase in fasting glucose with age.     

吸烟对脂质过氧化抗氧化系统及α1-抗胰蛋白酶的影响

通过检测３１３名吸烟血中自由基水平及α１－抗胰蛋白酶的变化。探讨氧自由基在吸烟致病中的作用。方法 试验分为非吸烟组１８９名，轻度吸烟组６８名，中度吸烟组１８０名；重度吸烟组６５名，采用硫代巴比妥酸法，放射免疫法，５‘５二硫代安息香酸直接法，Ｅｌｌｍａｎ法，酶标法分别检测脂质过氧化物，超氧化物歧化酶，谷胱甘肽过氧化物酶，谷胱甘肽及α１－ＡＴ。     

Substrate metabolism during modest hyperinsulinemia in response to isolated hyperketonemia in insulin-dependent diabetic subjects

To assess the metabolic effects of moderate hyperketonemia, six young male type 1 diabetic patients received a 200-minute intravenous (IV) infusion of (1) 0.9 mmol 3-hydroxybutyrate (3-OHB)/kg/h, and (2) saline. To ensure comparable metabolic conditions, a low-dose hyperinsulinemic euglycemic glucose clamp was performed from 5 hours before and throughout 3-OHB/saline infusions. The forearm technique was employed to estimate substrate fluxes in muscle. Infusion of 3-OHB caused: (1) increases (P less than .05) in circulating levels of 3-OHB (from 112 +/- 73 mumol/L to 825 +/- 111 mumol/L) and forearm arteriovenous differences of 3-OHB (from 19 +/- 10 mumol/L to 145 +/- 46 mumol/L), as well as an eightfold increase of plasma acetoacetate. (2) Decreased (P less than .05) levels of nonesterified fatty acids (NEFA; from 466 +/- 85 mumol/L to 201 +/- 14 mumol/L) and glycerol (from 39 +/- 7 mumol/L to 11 +/- 4 mumol/L) and decreased (P less than .05) arteriovenous differences of glycerol (from -16 +/- 8 mumol/L to -3 +/- 2 mumol/L). (3) Increased (P less than .05) levels of serum growth hormone (GH; from 4.1 +/- 1.5 micrograms/L to 15.9 +/- 8.0 micrograms/L). No change was recorded in circulating concentrations of free insulin, glucagon, glucose, lactate, or alanine. Nor were arteriovenous balances of these intermediary metabolites, isotopically determined glucose turnover or amounts of exogenously administered glucose affected. In conclusion, in type 1 diabetic man, the main regulatory effect of isolated hyperketonemia appears to be a direct negative feedback inhibition of lipolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 4,4'-di-isothiocyano-2,2'-stilbene disulphonate on iodide uptake by primary cultures of turtle thyroid follicular cells

Iodide uptake by primary cultures of turtle thyroid follicular cells is directly proportional to the Na+ concentration and is inversely proportional to the HCO3- concentration in culture medium, but is not affected by the Cl- concentration. Addition of 4,4'-di-isothiocyano-2,2'-stilbene disulphonate (DIDS; 10 mumol/l and higher doses) to medium containing different concentrations of Na+ (5-140 mmol/l), HCO3- (0-40 mmol/l) and Cl- (120 mmol/l) generally enhanced iodide uptake by the cultured cells; however, there was no significant effect in Na+-free and in low Cl- (90 mmol/l and less) medium. The inhibitory effects on iodide uptake of ouabain, frusemide and perchlorate were attenuated by DIDS which also antagonized the stimulatory effects on iodide uptake of TSH, although both DIDS and TSH increased the 125I- cell/medium ratio when they were given alone. At doses of 100 mumol/l and higher, DIDS lowered the intracellular pH of cultured cells when the pH of the medium was maintained at a constant level. It also increased the intracellular Cl- concentration, but had no effect on intracellular Na+ or K+. The input and specific resistances of cell membranes in cultured thyroid cells and in isolated thyroid slices increased (decreased conductance) after adding DIDS to the perfusion fluids. Both Na+/K+- and HCO3(-)-ATPase activities in homogenates of turtle thyroid tissue were inhibited by DIDS. Results from this investigation demonstrate (1) that in addition to preventing the leak of iodide from thyroid cells, DIDS may act to increase the sensitivity of the Na+-anion carrier to I- and thereby increases iodide uptake, and (2) that a HCO3(-)-Cl- exchange system is present in the thyroid cell membrane and appears to be linked to the transport of iodide into thyroid cells.     

Protein and RNA turnover in preterm infants and adults: a comparison based on urinary excretion of 3-methylhistidine and of modified one-way RNA catabolites

Urinary excretion of 3-methylhistidine in preterm infants (n = 42; 1,712 +/- 408 g, 4-91 days old) was 24.2 +/- 6 mumol/mmol creatinine or 2.26 +/- 0.56 mumol/kg body weight X day. In adults (n = 6; 66 +/- 10 kg, 17-50 years), the corresponding values were 10.5 +/- 1.1 mumol/mmol creatinine and 2.21 +/- 0.23 mumol/kg body weight X day. For both collectives, the breakdown per kg body weight of 3-methylhistidine-containing protein (i.e. actin and myosin) was similar, at approximately 0.7 g/kg X day (preterm infants 0.84, adults 0.60). Since the preterm infants studied contain approximately 21% muscle instead of the 43% found in adults, the 3-methylhistidine excretion in preterm infants probably indicates muscle (and intestinal) protein turnover to be about 3 times higher than in adults, a figure in accord with data on whole-body protein turnover in preterm infants and adults (approximately 15 g/kg X day and approximately 4 g/kg X day, respectively). Urinary excretion of pseudouridine (psi), 7-methylguanine (m7Gua) and N2, N2-dimethylguanosine (m2(2)G) can be used to estimate the turnover of rRNA, mRNA and tRNA, respectively. The values obtained (in mumol/mmol creatinine) in preterm infants are for psi: 164 +/- 32; for m7Gua: 39.1 +/- 9; and for m2(2)G: 10.6 +/- 2.1. In adults, the values are for psi: 25.3 +/- 3.1; for m7Gua: 4.8 +/- 0.89; and for m2(2)G: 1.53 +/- 0.38. This yields 3-4 times higher turnover rates in preterm infants than in adults for all 3 RNA classes: rRNA, 0.1 versus 0.038; tRNA, 1.87 versus 0.66; mRNA 2.35 versus 0.64 mumol/kg X day.     

Acid-base regulation of ion transport in rabbit ileum in vitro

Changes in acid-base balance have a major influence on ion transport in the ileum. The goals of the present study were to delineate (a) the specific transport processes most affected by changes in acid-base metabolism, (b) the individual roles of pH, PCO2, and concentration of HCO3- in modulating ion transport, and (c) the relationship between acid-base sensitive and other ion-transport systems. Ion transport and electrical parameters were measured in rabbit ilea in vitro under short-circuit conditions with systematic variations of pH, PCO2, and concentrations of HCO3-. Increasing HCO3- concentrations, with constant PCO2 and increasing pH, caused a decrease in electroneutral Na+ and Cl- absorption. At 5 mmol/L HCO3-, net fluxes of Na+ and Cl- were 5.9 +/- 1.4 and 4.5 +/- 1.1 microEq.cm-2.h-1, while at 50 mmol/L HCO3-, net Na+ and Cl- fluxes were 0.7 +/- 0.7 and 0.2 +/- 0.6 microEq.cm-2.h-1. Transepithelial HCO3(-)-gradient experiments suggested that serosal HCO3- was the principal factor. Changes in PCO2 showed a complex biphasic response, increasing net Cl- flux as PCO2 increased from 11-30 mm Hg in 5 mmol/L HCO3-; net Na+ flux increased as PCO2 was changed from 36 to greater than 100 mm Hg in 22 mmol/L HCO3-. In contrast, increasing pH in a bicarbonate-free N-2-hydroxyethylpiperazine-N'-2 = ethane sulfonic acid buffer did not significantly alter Na+ transport. Acid-base stimulated Na+ absorption was inhibited by 10(-3) mol/L amiloride, but not by bumetanide, consistent with the involvement of Na(+)-H+ exchange rather than Na(+)-Cl- cotransport. Epinephrine did not increase Na+ absorption under acid-base stimulated conditions, but glucose did, suggesting that the rate-limiting step for electroneutral absorptive processes under these conditions occurs at the apical rather than the basolateral membrane. Combining all experiments, a significant correlation existed between net flux of Na+ and HCO3- concentration (r = -0.72, P less than 0.05) and between net flux of Na+ and pH (r = -0.68, P less than 0.01). Chloride absorption was correlated with pH (r = 0.72, P less than 0.01). These results suggest a profound regulatory role for acid-base balance in electroneutral Na(+)-Cl- transport in rabbit ileum in vitro.     

Isolation, characterization, and attachment of rabbit distal colon epithelial cells.

The authors investigated various enzymatic digestion procedures for isolating epithelial cells from the distal colon of New Zealand White male rabbits. Rabbit mucosa was washed, diced, and digested for 90 minutes in one of five different solutions, including a new combination consisting of 0.03% collagenase IV and 0.1% pronase (solution V). Solution I (0.3% dispase) yielded 14.2 +/- 8.2 x 10(6) colonocytes/g mucosa, solution II (0.15% dispase and 0.03% collagenase) yielded 7.7 +/- 2.8 x 10(6) colonocytes/g mucosa, and solution III (0.03% collagenase IV) yielded 15.4 +/- 10(6) cells/g mucosa. Solutions I-III have previously been described for the isolation of colonocytes. Solution IV (0.1% pronase and 325 U/mL DNAase) was originally described for the isolation of nasal epithelial cells but yielded only 2.5 +/- 1.2 x 10(6) cells/g mucosa when applied to the isolation of colonocytes. The new combination of pronase and collagenase, solution V, yielded significantly more colonocytes, 34.5 +/- 3.0 x 10(6) cells/g mucosa, than previously described methods (P less than 0.01). Inclusion of 5 mmol/L ethylenediaminetetraacetic acid in any of the solutions enhanced neither viability nor yield. The digestion product of solution V could be enriched for crypts by serial low-speed centrifugations. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining for cytokeratins. Functional viability was tested by determining the presence of a Na+/H+ exchanger, using the pH fluorescent dye bis(carboxymethyl)-5(6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH. The authors document that sodium-dependent restoration of intracellular pH in colonocytes acid-loaded to a pH of 6.30 occurred at a rate of 0.19 +/- 0.02 pH U/min. Amiloride at concentrations of 1 mmol/L completely inhibited operation of the exchanger, as did sodium substitution with choline or tetramethylammonium. Lineweaver-Burke analysis at this intracellular pH showed a Michaelis constant of 10.71 mmol/L Na+ and a maximum velocity of 0.12 pH U/min. Exposing the colonocytes to 100 nmol/L phorbol 12,13-dibutyrate increased antiporter activity by 62.0%. Finally, the authors describe the synthesis of a new biomatrix composed of the basement membrane of 3T3 NIH fibroblasts that permits significantly improved colonocyte attachment than to glass, plastic, collagen types I or IV, or matrigel.     

Glucose and gluconeogenic substrate exchange by the forearm skeletal muscle in hyperglycemic and insulin-treated type II diabetic patients

To determine the contribution of skeletal muscle to fasting hyperglycemia in noninsulin dependent type II diabetes (NIDDM), the forearm balance of glucose, lactate, and alanine was quantified in 25 control subjects, 21 hyperglycemic (blood glucose: 11.6 mmol/L), and 19 insulin-treated patients with NIDDM (blood glucose: 5.8 mmol/L). Forearm glucose uptake was similar in controls (4.6 +/- 0.6 mumol L-1 min-1) and in hyperglycemic diabetic patients (4.5 +/- 0.9 mumol L-1 min-1). In spite of this, in the diabetic patients lactate (5.1 +/- 0.8 mumol L-1 min-1) and alanine (2.6 +/- 0.4) release by the forearm was 3- and 2-fold higher than in the control group (lactate: 1.7 +/- 0.8, P less than 0.005; and alanine: 1.3 +/- 0.2, P less than 0.05, respectively). The ratio of lactate release to glucose uptake was 57% and 18% in diabetic and control subjects, respectively. Insulin administration did not affect either glucose uptake or the release of gluconeogenic substrates by the forearm. It is concluded that: 1) in fasting patients with NIDDM, glucose is taken up by the skeletal muscle in normal amounts but preferentially used nonoxidatively with lactate formation. This suggests that, although the muscle does not contribute directly to fasting hyperglycemia, it may play an indirect role through an increased delivery of glucose precursors; and 2) insulin-induced normoglycemia is maintained by mechanisms that do not involve the exchange of glucose and gluconeogenic substrates by the skeletal muscle.     

Effect of gemfibrozil treatment in sulfonylurea-treated patients with noninsulin-dependent diabetes mellitus

This study was initiated to 1) assess gemfibrozil's ability to lower plasma triglyceride (TG) concentration in patients with NIDDM, and 2) determine whether this effect was associated with any changes in glycemic control. Measurements were made of mean hourly plasma glucose, insulin, TG, and FFA concentrations from 1200-1600 h in response to a test meal; hepatic glucose production (HGP); insulin-stimulated glucose uptake during a hyperinsulinemic glucose clamp study (MCR); and fasting plasma lipoprotein TG and cholesterol concentrations in 12 patients with NIDDM before and 3 months after gemfibrozil treatment. Although ambient plasma TG and FFA concentrations fell significantly, plasma glucose, insulin, HGP, concentrations fell significantly, plasma glucose, insulin, HGP, and glucose MCR did not change. However, when patients were divided into two groups, those with fasting plasma glucose levels above 9 mmol/L (fair control) and those with levels below 9 mmol/L (good control), a different phenomenon was observed. Patients in fair control had significant decreases in mean hourly plasma concentrations of glucose (15.1 +/- 1.7 to 12.6 +/- 0.9 mmol/L; P less than 0.001), insulin (523 +/- 59 to 471 +/- 75 pmol/L; P less than 0.001), FFA (652 +/- 150 to 504 +/- 76 mumol/L), and HGP (9.5 0.4 to 8.1 +/- 0.4 mumol/kg.min; P less than 0.005), and an increase in glucose MCR (2.63 +/- 0.49 to 3.72 +/- 0.54 mL/kg.min; P less than 0.07) in association with a fall in TG from 6.9 +/- 1.3 to 3.5 +/- 0.9 mmol/L (P less than 0.001). Although fasting low density lipoprotein cholesterol increased (1.8 +/- 0.2 to 2.7 +/- 0.2 mmol/L; P less than 0.05), the ratio of total to high density lipoprotein cholesterol decreased (6.84 +/- 0.88 to 5.80 +/- 1.05; P less than 0.02). Despite a significant fall in mean hourly TG concentration (4.6 +/- 0.7 to 3.8 +/- 0.7 mmol/L; P less than 0.001), neither insulin, FFA, HGP, nor glucose MCR changed in patients in good control. Furthermore, the mean hourly plasma glucose concentration increased from 9.2 +/- 0.7 to 11.7 +/- 1.4 mmol/L (P less than 0.001). Low density lipoprotein cholesterol also increased in this group (1.9 +/- 0.2 to 2.7 +/- 0.2 mmol/L; P less than 0.02), but, as before, the ratio of total to high density lipoprotein cholesterol decreased (8.15 +/- 1.93 to 6.36 +/- 1.03; P less than 0.02).     

Free fatty acids inhibit the glucose-stimulated increase of intramuscular glucose-6-phosphate concentration in humans

To test Randle's hypothesis we examined whether free fatty acids (FFAs) affect glucose-stimulated glucose transport/phosphorylation and allosteric mediators of muscle glucose metabolism under conditions of fasting peripheral insulinemia. Seven healthy men were studied during somatostatin-glucose-insulin clamp tests [plasma insulin, 50 pmol/L; plasma glucose, 5 mmol/L (0-180 min), 10 mmol/L (180-300 min)] in the presence of low (0.05 mmol/L) and increased (2.6 mmol/L) plasma FFA concentrations. (31)P and (1)H nuclear magnetic resonance spectroscopy was used to determine intracellular concentrations of glucose-6-phosphate (G6P), inorganic phosphate, phosphocreatine, ADP, pH, and intramyocellular lipids. Rates of glucose turnover were measured using D-[6,6-(2)H(2)]glucose. Plasma FFA elevation reduced rates of glucose uptake at the end of the euglycemic period (R(d 150-180 min): 8.6 +/- 0.5 vs. 12.6 +/- 1.6 micromol/kg.min, P < 0.05) and during hyperglycemia (R(d 270-300 min): 9.9 +/- 0.6 vs. 22.3 +/- 1.7 micromol/kg.min, P < 0.01). Similarly, intramuscular G6P was lower at the end of both euglycemic (G6P(167-180 min): -22 +/- 7 vs. +24 +/- 7 micromol/L, P < 0.05) and hyperglycemic periods (G6P(287-300 min): -7 +/- 9 vs. +28 +/- 7 micromol/L, P < 0.05). Changes in intracellular inorganic phosphate exhibited a similar pattern, whereas FFA did not affect phosphocreatine, ADP, pH, and intramyocellular lipid contents. In conclusion, the lack of an increase in muscular G6P along with reduction of whole body glucose clearance indicates that FFA might directly inhibit glucose transport/phosphorylation in skeletal muscle.     

糖皮素激素及茶碱对哮喘小鼠气道炎症与肺内一氧化氮抑？…

目的 探讨糖皮质激素、茶碱对哮喘气道炎症和肺内一氧化氮（ＮＯ）水平、诱生型一氧化氮合酶（ｉＮＯＳ）表达的影响。方法 ＢＡＬＢ／ｃ小鼠分为６组：哮喘模型组（Ａ，１０只），正常对照组（Ｎ，１０只），地塞米松雾化吸入组（Ｃ１，７只），地塞米松腹腔注射组（Ｃ２，７只），布地奈德气雾剂组（Ｃ３，７只），茶碱组（Ｔ，１０只）。建立哮喘模型，给予糖皮质激素、氨茶碱、观察支气管肺泡灌洗液（ＢＡＬＦ）中白细胞总数、     

Uptake of L-carnitine by a human intestinal epithelial cell line, Caco- 2

BACKGROUND & AIMS: The mechanism of intestinal uptake of L-carnitine is controversial. The aim of this study was to clarify the mechanism and regulation of L-carnitine uptake. METHODS: Uptake of [3H]-L-carnitine was measured across the apical membrane of confluent monolayers of Caco- 2 cells. RESULTS: [3H]-L-carnitine uptake was linear and appreciable for up to 7 minutes with minimal metabolic alteration, was temperature- and Na(+)-(but not pH-) dependent, and included a saturable component with an apparent Michaelis constant of 45.5 +/- 6.5 mumol/L and a maximum velocity of 83.5 +/- 5.6 nmol.mg protein-1.5 min-1. Unlabeled L- carnitine and its structurally related analogues significantly (P < 0.01) inhibited [3H]-L-carnitine uptake, whereas unrelated compounds were ineffective. L-Carnitine uptake was also energy-dependent, being significantly (P < 0.01) inhibited by metabolic inhibitors. Our results also suggested that a calmodulin- but not a protein kinase C- or protein kinase A-mediated pathway plays a role in regulating L- carnitine uptake by Caco-2 cells. CONCLUSIONS: L-carnitine uptake by intestinal epithelial cells (Caco-2) involves a carrier-mediated system that is temperature-, Na(+)-, and energy-dependent and seems to be under the regulation of a calmodulin-mediated pathway. (Gastroenterology 1996 Dec;111(6):1534-40)     

The physiologic action of insulin on glucose uptake and its relevance to the interpretation of the metabolic clearance rate of glucose

Glucose uptake (Ru) is dependent upon the concentrations of both glucose and insulin. The metabolic clearance rate of glucose (MCRG), has been used as an in vivo measure of insulin action, because it was said to be independent of the prevailing glucose concentration. The validity of this assumption has recently been challenged. In this study, the effect of insulin concentration on the rate of glucose uptake (Ru) and on the MCRG was studied during euglycemia (5.1 +/- 0.3 mmol/L) and moderate hyperglycemia (10.4 +/- 0.5 mmol/L) in 17 experiments on nine normal ambulant volunteers. Stable plasma insulin levels were maintained with fixed infusion rates of insulin (0-300 mU/kg/h) and somatostatin (7.5 micrograms/min). At low insulin concentrations (less than 5 microU/mL) the increase in glucose uptake in response to hyperglycemia was small (5.3 +/- 2.3 mumol/kg/min). In contrast, with insulin levels more than 25 microU/mL, there was a steep rise in glucose uptake with hyperglycemia (55 +/- 3 mumol/kg/min; range: 44-74 mumol/kg/min). The metabolic clearance rate of glucose fell by an average of 32% with hyperglycemia in the studies at the lowest insulin levels (2.2 +/- 0.6 v 1.5 +/- 0.1 mL/kg/min; 0.15 greater than P greater than 0.1). There was no change in the MCRG in the subjects studied at higher insulin levels. It is concluded that (1) low concentrations of insulin are essential for the increase in glucose disposal during hyperglycemia; and (2) provided insulin levels are more than 25 microU/mL and plasma glucose less than 11 mmol/L, MCRG is independent of the plasma glucose concentration and is therefore a valid measure of insulin-mediated glucose uptake.     

Role of cyclic nucleotides in rapid platelet adhesion to collagen

Adhesion of human platelets to type I collagen under arterial flow conditions is extremely fast, being mediated primarily by the alpha 2 beta 1 integrin (glycoprotein Ia/IIa). We have investigated the involvement of cyclic nucleotides in platelet adhesion to soluble native collagen immobilized on Sepharose beads using a new microadhesion assay under arterial flow conditions. To prevent platelet stimulation by thromboxanes and adenosine diphosphate (ADP), experiments were performed with aspirin-treated platelets in the presence of ADP-removing enzyme systems such as creatine phosphate/creatine phosphokinase or apyrase. Rapid reciprocal changes in platelet adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) occurred during adhesion. cAMP levels in adherent platelets were 2.4-fold lower than in effluent platelets or in static controls, whereas cGMP levels were increased 2.4-fold. These results suggest that contact between platelets and collagen stimulates guanylate cyclase and inhibits adenylate cyclase. This occurs in the absence of the platelet release reaction. We also studied short-term effects of agents that regulate cyclic nucleotide synthesis, prostaglandin E1 (PGE1) and sodium nitroprusside (SNP). After only 3.8 seconds at 10 to 30 dyne/cm2, PGE1 (10 mumol/L) increased cAMP 16.4- fold, whereas SNP (50 mumol/L) increased cGMP ninefold and caused a 3.2- fold increase in cAMP. Both PGE1 and SNP rapidly (< 5 seconds) inhibited platelet adhesion in a dose-dependent manner that was correlated with the increase in cyclic nucleotides. Our data suggest that cAMP and cGMP play a regulatory role in the initial phases of platelet adhesion to collagen mediated by the alpha 2 beta 1 integrin receptor.     

Plasma L-arginine and metabolites of nitric oxide synthase in patients with left-to-right shunt after intracardiac repair

STUDY OBJECTIVE: Human plasma L-arginine serves as a substrate pool for endothelial-derived nitric oxide (NO) synthase. In this pilot study, we tested the hypothesis that plasma L-arginine and other metabolites of the L-arginine NO pathway could correlate with postoperative pulmonary hypertension after cardiopulmonary bypass (CPB). DESIGN: Forty-two patients (median age, 0.5 years; range, 0.1 to 28 years) with atrial septal defect (n = 15), ventricular septal defect (n = 18), atrioventricular canal (n = 8), and aortopulmonary window (n = 1) were enrolled. The influence of patient age, preoperative pulmonary hypertension, duration of CPB, plasma L-arginine, guanosine 3', 5'-cyclic monophosphate (cGMP), and nitrate on postoperative pulmonary hypertension during the first 24 h after CPB was studied by logistic regression. RESULTS: Nineteen of 42 patients were found to have preoperative pulmonary hypertension. Thirteen of 42 patients showed persistent pulmonary hypertension after intracardiac repair with a mean pulmonary artery pressure (PAP) of 38 mm Hg (range, 23 to 55 mm Hg) at 24 h after CPB. L-arginine concentrations in plasma were significantly lower 24 h after CPB than before: 52 mumol/L (range, 18 to 95 mumol/L) vs 79 mumol/L (range, 31 to 157 mumol/L). Plasma cGMP levels were higher and plasma nitrate levels were lower immediately after weaning from CPB (p < 0.0033). On logistic regression analysis, only patient age (p = 0.02) and preoperative PAP (p = 0.01) were related to postoperative pulmonary hypertension. CONCLUSION: Low plasma L-arginine does not relate to persistent pulmonary hypertension in patients with left-to-right shunt after CPB and intracardiac repair.