Short-term regulation of multidrug resistance-associated protein 3 in rat and human hepatocytes

The short-term regulation of multidrug resistance-associated protein 3 (Mrp3/MRP3) by cAMP and PKC was investigated in sandwich-cultured rat and human hepatocytes and isolated perfused rat livers. The modulator glucagon (500 nM) and the phorbol ester PMA (0.1 muM) were utilized to increase intracellular cAMP and PKC levels, respectively. In glucagon-treated rat hepatocytes, efflux of the Mrp3 substrate 5-(6)-carboxy-2',7'-dichlorofluorescein (CDF) increased approximately 1.5-fold, even in hepatocytes treated with the organic anion transporter (Oatp) inhibitor sulfobromophthalein (BSP). Confocal microscopy revealed more concentrated Mrp3 fluorescence in the basolateral membrane (less diffuse staining pattern) with glucagon treatment. PMA had no effect on Mrp3 activity or localization in sandwich-cultured rat hepatocytes. Glucagon and PMA treatment in isolated perfused rat livers resulted in a threefold increase (14 +/- 4.6 mul.min(-1).g liver(-1)) and a fourfold decrease (1.3 +/- 0.3 mul.min(-1).g liver(-1)) in CDF basolateral clearance compared with control livers (4.7 +/- 2.3 mul.min(-1).g liver(-1)), whereas CDF biliary clearance was not statistically different. In sandwich-cultured human hepatocytes, glucagon treatment resulted in a 1.3-fold increase in CDF efflux and a concomitant increase in MRP3 fluorescence in the basolateral membrane. In summary, cAMP and PKC appear to be involved in the short-term regulation of Mrp3/MRP3, as demonstrated by alterations in activity and localization in rat and human hepatocytes.     

STEADY STATE MAINTENANCE OF ELECTROLYTES IN THE SPINAL CORD OF THE FROG

Isolated frog spinal cords equilibrated from 3 to 24 h in Ringer's solution maintained steady state conditions with regard to electrolyte composition. Total sodium and chloride contents measured on the same spinal cords were found to be nearly equivalent (Na = 46.6 ± 1.4μmol/g wet wt vs Cl = 46.2 ± 1.2μmol/g wet wt). Calcium and magnesium contents were 3.0 ± 0.5 and 4.8 ± 0.2pμol/g wet wt respectively for fresh spinal cords and 2.1 ± 0.4 and 5.5 ± 0.9pmol/g wet wt respectively for spinal cords equilibrated for 24 h. Zinc content was 0.29 ± 0.01 μmol/g wet wt. Insulin space was found to be smaller than sucrose space (0.24 ml/g vs 0.37 ml/g). Sodium and chloride spaces were slightly higher than sucrose space. Sodium and chloride in the non-sucrose space was 4.8 and 9.5 μmol/g wet wt. Residual radioactive sodium, or sodium content of spinal cords washed out for 180 min in non-radioactive Ringer's solution or in choline or lithium Ringer's solution, was 4.2 ± 0.4μmol/g wet wt (n = 9). The agreement between residual sodium content and sodium in the non-sucrose space suggests that the mean intracellular sodium content of the spinal cord neurons is low.     

Calcium metabolism in rat hepatocytes.

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.     

Cytochrome P-450 levels and EROD activity in the Adriatic sturgeon (Acipenser naccarii, Chondrostei) and its induction by β-Naphthoflavone

The cytochrome P-450 enzyme system was studied in juvenile Acipenser naccarii (317.6±77 g initial live weight) reared at 23°C and at a standard diet over a period of two month before using for experiments (final weight 546.3±148.8 g). While determining normal levels of cytochrome P-450 in liver tissue its induction was also studied under exposure to P-naphthoflavone (BNF; single dose at 80 mg/Kg body weight 52 hrs prior to sampling). Control fish showed total cytochrome P-450 levels in liver microsomes around 0.40±0.12 nmoles/mg proteins (n=12) while in BNF exposed fish the values increased significantly (0.55±0.11nmoles/mg proteins; n=12). Ethoxyresorufin-O-deethylase (EROD) in control fish was 0.015±0.02 nmoles/mg proteins/min (n=12); it was found to be NADPH dependent and inducible by BNF. Western Immunoblotting and enzyme-linked-immunosorbent (ELISA) analyses of liver microsomes from untreated and BNF-exposed fish, using an antibody to rat cytochrome P-450 1A1 isoform (P-450 1A1) (Nelson et al.,1996) suggested the presence of a constitutive and BNF-inducible cytochrome P-450 homologous to rat P-450 protein.
Measurement of cytochrome P-450-dependent EROD activity and P-450 1A1 expression levels in A. naccarii may be a useful parameter in monitoring environmental pollution.     

Zea mays L. extracts modify glomerular function and potassium urinary excretion in conscious rats

Diuretic and uricosuric properties have traditionally been attributed to corn silk, stigma/style of Zea mays L. Although the diuretic effect was confirmed, studies of the plant's effects on renal function or solute excretion were lacking. Thus, we studied the effects of corn silk aqueous extract on the urinary excretion of water, Na+, K+, and uric acid. Glomerular and proximal tubular function and Na+ tubular handling were also studied. Conscious, unrestrained adult male rats were housed in individual metabolic cages (IMC) with continuous urine collection for 5 and 3 h, following two protocols. The effects of 25, 50, 200, 350, and 500 mg/kg body wt. corn silk extract on urine volume plus Na+ and K+ excretions were studied in water-loaded conscious rats (2.5 ml/100 g body wt.) in the IMC for 5 h (Protocol 1). Kaliuresis was observed with doses of 350 (100.42 +/- 22.32-120.28 +/- 19.70 microEq/5 h/100 g body wt.; n = 13) and 500 mg/kg body wt. (94.97+/- 29.30-134.32 +/- 39.98 microEq/5h/100 g body wt.; n = 12; p<0.01), and the latter dose resulted in diuresis as well (1.98 +/- 0.44-2.41 +/- 0.41 ml/5 h/100 g body wt.; n = 12; p<0.05). The effects of a 500 mg/kg body wt. dose of corn silk extract on urine volume, Na+, K+ and uric acid excretions, and glomerular and proximal tubular function, were measured respectively by creatinine (Cler) and Li+ (ClLi) clearances and Na+ tubular handling, in water-loaded rats (5 ml/100 g body wt.) in the IMC for 3 h (Protocol 2). Clcr (294.6 +/- 73.2, n = 12, to 241.7 +/- 48.0 microl/ min/100 g body wt.; n = 13; p<0.05) and the Na+ filtered load (41.9 +/- 10.3, n = 12, to 34.3 +/- .8, n = 13, p<0.05) decreased and ClLi and Na+ excretion were unchanged, while K+ excretion (0.1044 +/- 0.0458, n=12, to 0.2289 +/- 0.0583 microEq/min/100 body wt.; n = 13; p<0.001) increased. For Na+ tubular handling, the fractional proximal tubular reabsorption (91.5 +/- 3.5, n = 12, to 87.5 +/- 3.4%; n = 13; p<0.01) decreased, and both fractional distal reabsorptions--I and II--increased (96.5 +/- 1.5, n = 12, to 97.8 +/- 0.9%; n = 13; p<0.01; and 8.2 +/- 3.5, n = 12, to 12.2 +/- 3.4%, n = 13, p<0.01, respectively). To summarize, in water-loaded conscious rats (2.5 ml/100 body wt.), corn silk aqueous extract is diuretic at a dose of 500 mg/kg body wt. and kaliuretic at doses of 350 and 500 mg/kg body wt. In water-loaded conscious rats (5.0 ml/100 g body wt.), corn silk aqueous extract is kaliuretic at a dose of 500 mg/kg body wt., but glomerular filtration and filtered load decrease without affecting proximal tubular function, Na+, or uric acid excretion.     

Glutamate uptake in primary cultures of biliary epithelial cells from normal rat liver

Biliary epithelial cells (BEC) were isolated from normal rat liver with high purity (> 95%) as revealed by morphological criteria as well as staining for gamma-glutamyl transferase and cytokeratin 19. During cultivation for 96 hr flattening of the cells and a loss of microvilli was apparent, while the cytokeratin 19-positive phenotype was maintained. The BEC contained a sodium-dependent as well as a sodium-independent uptake system for glutamate with high capacity. Both activities increased transiently during cultivation peaking after 72 and 48 hr, respectively. After 72 hr, apparent kinetic constants could be calculated for the sodium dependent (K_m = 13.6 mM; V_max = 388 nmoles/min/mg protein) and for the sodium-independent system. (K_m = 10.8 mM; V_max = 132 nmoles/min/mg protein). The transient increase of both transport systems was suppressed by dexamethasone. The sodium-dependence showed a threshold concentration of about 35 mM sodium. Inhibition by kainate was much less potent for BEC than for hepatocytes. These data indicate that BEC contain transport systems for glutamate different from those in hepatocytes and which may be involved in the intrahepatic reabsorbtion of glutamate from bile.Abbreviations BEC biliary epithelial cells - DMEM Dulbecco's Modified Eagle's Medium - GGT gamma-glutamyl transferase - Dex dexamethasone - Glu glutamate - N-Me-AIB N-methyl-aminoisobutyrate - Hep hepatocytes - FBS Fetal bovine serum     

Glomerular filtration rate determinations in conscious type II diabetic mice

Diabetic nephropathy is a major cause of end-stage renal disease worldwide. The current studies were performed to determine the later stages of the progression of renal disease in type II diabetic mice (BKS; db/db). Methodology was developed for determining glomerular filtration rate (GFR) in conscious, chronically instrumented mice using continuous intravenous infusion of FITC-labeled inulin to achieve a steady-state plasma inulin concentration. Obese diabetic mice exhibited increased GFR compared with control mice. GFR averaged 0.313 ± 0.018 and 0.278 ± 0.007 ml/min in 18-wk-old obese diabetic (n = 11) and control (n = 13) mice, respectively (P < 0.05). In 28-wk-old obese diabetic (n = 10) and control (n = 15) mice, GFR averaged 0.348 ± 0.030 and 0.279 ± 0.009 ml/min, respectively (P < 0.05). GFR expressed per gram BW was significantly reduced in 18- and 28-wk-old obese diabetic compared with control mice (5.9 ± 0.3 vs. 9.0 ± 0.3; 6.6 ± 0.6 vs. 7.8 ± 0.3 μl·min(-1)·g body wt(-1)), respectively (P < 0.05). However, older nonobese type II diabetic mice had significantly reduced GFR (0.179 ± 0.023 ml/min; n = 6) and elevated urinary albumin excretion (811 ± 127 μg/day) compared with obese diabetic and control mice (514 ± 54, 171 ± 18 μg/day), which are consistent with the advanced stages of renal disease. These studies suggest that hyperfiltration contributes to the progression of renal disease in type II diabetic mice.     

Quantitative analysis of internalization of glucagon by isolated hepatocytes

Biochemical methods have been used to quantitate total, acid-stable and acid-labile association of (mono[125I]iodoTyr10) glucagon with rat hepatocytes in suspension to evaluate internalization of glucagon and its receptors. Internalization is inhibited by low temperature, phenylarsine oxide, and by blocking receptor binding, consistent with receptor-mediated endocytosis. Approximately 30% of the total cell-associated hormone is internalized at 30 min of incubation. The rate declines until 90 min when the internalization of glucagon ceases, although the cells remain competent to internalize asialofetuin. From 90 min to 4 h, 27% of the maximum label internalized at 30 min remains within cells. The number of cell surface receptors decreases but the affinity of those remaining is unchanged. However, 1.7-2.7 surface receptors are lost to binding for each molecule of radiolabeled glucagon internalized. Uptake occurs according to a rate constant of 0.183 min-1 (t1/2 = 3.8 min). We conclude that (i) hepatocytes internalize a finite quantity of glucagon, implying the existence of undefined regulatory mechanisms; (ii) hormone is retained for greater than 2 h within cells and may play a physiological role within cells; and (iii) both occupied and unoccupied receptors become inaccessible to extracellular hormone as internalization proceeds; rapid recycling of receptors does not occur.     

Plasma glucagon and free fatty acid responses to a glucose load in the obese spontaneous hypertensive rat (SHROB) model of metabolic syndrome X

Metabolic Syndrome X is a cluster of abnormalities including insulin resistance, hyperlipidemia, hypertension, and obesity. We sought to determine if excess plasma glucagon and free fatty acids (FFA) might contribute to the insulin resistance in the obese spontaneous hypertensive rat (SHROB), a unique animal model of leptin resistance and metabolic Syndrome X. SHROB were extremely hyperinsulinemic and mildly glucose intolerant compared with lean SHR. SHROB had elevated fasting plasma glucagon and FFA, and showed paradoxical responses to an oral glucose challenge, with increased glucagon at 30 and 60 min postchallenge (200% plus minus 45% and 91% plus minus 13%, respectively; n = 9). In lean SHR, glucagon was nearly unchanged by glucose loading (<30% increase, P > 0.05; n = 5). Plasma FFA were not affected by a glucose load in SHROB, whereas SHR showed a decrease of 40% plus minus 6% (n = 5--9). The I/G molar ratio changed in opposite directions in the two genotypes, with a decrease in SHROB at 30 and 60 min, in contrast to the appropriate increase at 30 and 60 min postchallenge in the lean SHR (P < 0.01; n = 5--9). Administration of 500 ng/kg exogenous glucagon to SHR raised glucagon 56% plus minus 5% to a level that was similar to fasting SHROB. This level of circulating glucagon was sufficient to elevate glucose and insulin during the 7 hr of observation (n = 9). Based on these results, we suggest that fasting hyperglucagonemia and impaired suppression of glucagon secretion and FFA in response to an oral glucose load may contribute to insulin resistance and glucose intolerance in the SHROB model of metabolic Syndrome X.     

Utilisation of triacylglycerol and non-esterified fatty acid by the working rat heart: myocardial lipid substrate preference

Utilisation and subsequent metabolic fate (oxidation; tissue lipid deposition) of non-esterified fatty acid (NEFA), very-low-density lipoprotein-triacylglycerol (VLDL-TAG), and chylomicron-triacylglycerol (CM-TAG) alone or in combination by isolated working rat heart were examined. Cardiac mechanical function was maintained regardless of lipid substrate used. NEFA and CM-TAG were assimilated to a greater extent than VLDL-TAG; CM-TAG utilisation (76+/-10 nmol fatty acid/min per g wet wt.; n=8), but not VLDL-TAG utilisation (16+/-2 nmol fatty acid/min per g wet wt.; n=8), was suppressed in the presence of NEFA, but TAG (CM or VLDL) did not alter NEFA utilisation (57+/-9 nmol fatty acid/min per g wet wt.; n=8). Most (about 75%) of the lipid utilised was oxidised. In the presence of NEFA, CM-TAG deposition as tissue lipid was preserved, despite decreased CM-TAG oxidation; metabolic fate of VLDL-TAG was unaffected by NEFA. TAG (CM or VLDL) in the perfusate tended to decrease lipoprotein lipase (LPL) activity; this may be a reflection of increased LPL turnover in the presence of lipoproteins.     

Adsorption of bile salts by the cestodes, Hymenolepis diminuta and H. microstoma.

The accumulation of purified sodium taurocholate (NaTC) and sodium glycocholate (NaGC) by Hymenolepis diminuta and Hymenolepis microstoma (Cestoda: Cyclophyllidea) was determined using radioactive bile salts. H. diminuta reached equilibrium levels of approximately 120 nmoles NaTC/g dry wt and 300 nmoles NaGC/g dry wt. Presentation of the bile salts in mixed micelles with 0.35 mM oleic acid did not alter these values. With H. microstoma, the maxima were 195 nmoles NaTC/g dry wt and 614 nmoles NaCG/g dry wt. These values were similarly unaffected by the addition of 0.35 mM oleic acid to the micelles. Equilibrium values of this magnitude, in media containing as much as 25 or 30 mM bile salt, and the maintenance of this level during incubations of 15 to 60 min eliminated the possibility that the accumulation was by diffusion or by any form of mediated transport into the worm. The accumulation on NaTC by H. diminuta was [Na+] independent, and insensitive to ouabain, DNP, and high [K+]. These observations, the maintenance of different levels of NaTC and NaGC, and the failure of the 2 bile salts to compete indicated that there was no active excretion mechanism operating in a fashion similar to the active transport of bile salts in the vertebrate small intestine. It was concluded that the accumulation of NaTC by H. diminuta was actually adsorption to the tegument. Comparable, although more limited, experiments extended this conclusion to the accumulation of NaGC by H. diminuta and of NaTC and NaGC by H. microstoma. It is suggested that bile salt monomers, rather than intact micelles, adsorb to specific loci on the tegument.     

Phase I and II metabolism and carbohydrate metabolism in cultured cryopreserved porcine hepatocytes

Primary porcine hepatocytes were cryopreserved using freezing boxes or a programmable freezer (PF). Upon thawing and culturing in 12-well plates cryopreserved hepatocytes were compared with their fresh controls on days 1 and 2 after plating. Cryopreserved hepatocytes attached approximately as well as fresh hepatocytes and useful cultures were obtained. In cryopreserved hepatocytes, coumarin 7-hydroxylation, 6beta-testosterone hydroxylation and p-nitrophenol glucuronidation were reduced to about 10-40, 35 and 40%, respectively, compared to their fresh counterparts. Glycogen synthesis in cryopreserved hepatocytes was reduced to about 30% on day 1 of culture and about 47% on day 2 of culture compared to the synthesis in fresh hepatocytes. Both fresh and cryopreserved hepatocytes increased the synthesis by twofold in response to stimulation with insulin. Reduced basal levels of glycogen and of glycogen synthesis could be explained by an increased energy demand in cryopreserved hepatocytes needing to repair damages caused by cryopreservation. Glycogenolysis was reduced to about 50% in cryopreserved hepatocytes and gluconeogenesis to about 40% of the glucose production in fresh hepatocytes. In both fresh and cryopreserved hepatocytes the glucose production from glycogenolysis and gluconeogenesis, respectively, was increased fourfold in response to stimulation with glucagon. Overall, the hepatocytes cryopreserved in boxes had a tendency to perform better than hepatocytes cryopreserved in a programmable freezer. In conclusion, the cryopreserved hepatocytes were metabolic active; however, to a lower extent than the fresh hepatocytes, although, the cryopreserved hepatocytes responded as well as the fresh hepatocytes to insulin and glucagon.     

Static contraction increases arachidonic acid levels in gastrocnemius muscles of cats

Static contraction of hind-limb muscles is well known to increase reflexly cardiovascular function. Recently, blockade of cyclooxygenase activity has been reported to attenuate the reflex pressor response to contraction, a finding which suggests that working skeletal muscle releases arachidonic acid metabolites. Therefore, we measured the effects of static contraction and ischemia on arachidonic acid levels in the gastrocnemius muscles of barbiturate-anesthetized cats treated with indomethacin. Unesterified arachidonic acid levels were measured by high-pressure liquid chromatography. We found that static contraction of freely perfused gastrocnemius muscles increased arachidonic acid levels from 4.4 +/- 1.0 to 10.3 +/- 2.2 nmol/g wet wt (n = 12; P less than 0.005). Likewise, static contraction of gastrocnemius muscles made ischemic for 2 min before the onset of the contraction period increased arachidonic acid levels from 12.6 +/- 2.3 to 21.0 +/- 2.0 nmol/g wet wt (n = 12; P less than 0.01). Lastly, 2 min of ischemia with the gastrocnemius muscles at rest increased arachidonic acid levels from 5.9 +/- 1.1 to 10.5 +/- 3.0 nmol/g wet wt (n = 18; P less than 0.02). We conclude that both static contraction and ischemia increase arachidonic acid levels in working hindlimb muscle.     

Downhill running: a model of exercise hyperemia in the rat spinotrapezius muscle.

To utilize the rat spinotrapezius muscle as a model to investigate the microcirculatory consequences of exercise training, it is necessary to design an exercise protocol that recruits this muscle. There is evidence that the spinotrapezius is derecruited during standard treadmill exercise protocols performed on the uphill treadmill (i.e., 6 degrees incline). This investigation tested the hypothesis that downhill running would effectively recruit the spinotrapezius muscle as assessed by the presence of an exercise hyperemia response. We used radioactive 15-microm microspheres to determine blood flows in the spinotrapezius and selected hindlimb muscles of female Sprague-Dawley rats at rest and during downhill (i.e., -14 degrees incline; 331 +/- 5 g body wt, n = 7) and level (i.e., 0 degrees incline; 320 +/- 11 g body wt, n = 5) running at 30 m/min. Both level and downhill exercise increased blood flow to all hindlimb muscles (P < 0.01). However, in marked contrast to the absence of a hyperemic response to level running, blood flow to the spinotrapezius muscle increased from 26 +/- 6 ml.min(-1).100 g(-1) at rest to 69 +/- 8 ml.min(-1).100 g(-1) during downhill running (P < 0.01). These findings indicate that downhill running represents an exercise paradigm that recruits the spinotrapezius muscle and thereby constitutes a tenable physiological model for investigating the adaptations induced by exercise training (i.e., the mechanisms of altered microcirculatory control by transmission light microscopy).     

The interaction of glucagon, gastric inhibitory peptide and somatostatin with cyclic AMP production systems present in rat gastric glands

The effects of glucagon, gastric inhibitory peptide (GIP) and somatostatin on the generation of cyclic AMP have been studied under basal and histamine- or secretin-stimulated conditions in tubular gastric glands isolated by means of EDTA from the rat fundus and antrum. Four types of cell could be identified by electron microscopy; namely, parietal, mucous, peptic and some endocrine cells with a good morphological preservation of the cellular topography as seen in the intact mucosa. Immunoreactive somatostatin was found in antral glands (210 +/- 16 ng/g cell, wet wt., n = 9) as well as in fundic glands, but in smaller concentration (50 +/- 8 ng/g cell, wet wt., n = 9). (1) In rat fundic glands, glucagon, in supraphysiologic doses (3 . 10(-9) -5 . 10(-7) M), raised cyclic AMP levels 46 times above the basal. At maximally effective doses, combination of glucagon plus histamine was not additive whereas glucagon and secretin stimulations resulted in an additive response. Somatostatin (10(-10) -10(-7) M) inhibited both glucagon- and histamine-induced cyclic AMP production, whereas cimetidine specifically blocked the histaminergic stimulation. (2) In the same conditions, 10(-6)M glucagon produced a marginal effect (4-fold increase) in rat antrum, whereas GIP (10(-9) -10(-6)M) was unable to induce a significant rise of cyclic AMP production in either fundic or antral glands, or to prevent cyclic AMP production stimulated by histamine. (3) The present data do not support the view that circulating glucagon or GIP may regulate gastric secretion directly by a cyclic AMP-dependent mechanism in rat gastric glands and raise the possibility that gastric somatostatin may be the final mediator of the inhibitory actions of these hormones on acid secretion. (4) It is proposed that pancreatic glucagon acts through a receptor-cyclic AMP system which is specific for the bioactive peptide enteroglucagon ('oxyntomodulin'), probably in rat parietal cells.     

Acetyl coenzyme A concentrations in plant tissues

Despite the importance of acetyl coenzyme A in many facets of metabolism and the availability of methods for estimation of its concentration, data for acetyl-CoA concentrations in plant tissues have been very scarce. A method using reversed phase HPLC for the quantitative estimation of acetyl-CoA was applied to a variety of plant tissues. In three different developing oilseeds the bulk acetyl-CoA concentration ranged from 5 to 25 nmoles/g fresh weight. In Arabidopsis thaliana leaves it was 5 nmoles/g fresh weight, and in Spinacia oleracea leaves 6.8 nmoles/g fresh weight. Immediate quenching of the harvested tissue in liquid nitrogen is needed to obtain high recoveries of acetyl-CoA.     

Oxyntomodulin (glicentin-(33-69)): pharmacokinetics, binding to liver cell membranes, effects on isolated perfused pig pancreas, and secretion from isolated perfused lower small intestine of pigs

The pharmacokinetics of purified synthetic oxyntomodulin were studied after infusing it into euglycaemic pigs at two rates. The elimination of the peptide from plasma was characterized by two components, a fast one (t1/2 7.2 +/- 0.6 min) and a slow one (t1/2 20.4 +/- 3.8 min) (mean +/- S.E.M., n = 7). The metabolic clearance rate was independent of infusion rate (6.96 +/- 0.99 vs 7.44 +/- 0.98 ml/kg . min (mean +/- S.E.M., n = 7). The synthetic peptide bound to pig hepatic glucagon receptors, but with approximately 2% of the affinity of glucagon, and showed insulinotropic and somatostatinotropic effects when infused into isolated perfused pig pancreases at concentrations higher than 10(-10) M. A dose-dependent increase was also shown for pancreatic glucagon output. A naturally occurring peptide, identified as oxyntomodulin by gel filtration and HPLC, was released into the circulation from the pig lower small intestinal mucosa upon intraluminal administration of glucose, and represented 25 +/- 3.8% of the secreted glucagon-like immunoreactivity. 11 +/- 2.3% of the secreted glucagon-like immunoreactivity was indistinguishable from glucagon itself upon gel filtration; thus at least 36% of the glucagon-like immunoreactivity secreted from the intestinal mucosa is already in an active form.     

Characteristics of butanol metabolism in alcohol dehydrogenase-deficient deermice.

Deermice lacking the low-Km alcohol dehydrogenase eliminated butan-1-ol, a substrate for microsomal oxidation but not for catalase, at 117 mumol/min per kg body wt. Microsomal fractions and hepatocytes metabolized butan-1-ol also (Vmax. = 6.7 nmol/min per nmol of cytochrome P-450, Km = 0.85 mM; Vmax. = 5.3 nmol/min per 10(6) cells, Km = 0.71 mM respectively). These results are consistent with alcohol oxidation by the microsomal system in these deermice.     

Antioxidants attenuate endotoxin-induced microvascular leakage of macromolecules in vivo.

The purpose of this study was to examine whether antioxidants attenuate endotoxin-induced microvascular hyper-permeability for macromolecules in the hamster cheek pouch. Twenty-two adult male Syrian hamsters were anesthetized, and a removable plastic chamber was placed in the cheek pouch to observe and collect suffusate from the microvasculature. Fluorescent-labeled dextran (FITC-D; mol wt 150,000) was injected intravenously, and changes in the number of microvascular leaky sites and microvascular clearance of FITC-D were measured in five groups: saline control (group 1, n = 4), endotoxin (0.1 mg/ml) suffusion for 120 min (group 2, n = 6), endotoxin plus dimethyl sulfoxide (1.0 g/kg iv; group 3, n = 4), endotoxin plus allopurinol (30 mg/kg ip; group 4, n = 4), and endotoxin plus dimethyl sulfoxide and allopurinol (group 5, n = 4). The number of leaky sites and the FITC-D clearance were significantly higher in group 2 [45 +/- 18 (SD) sites/cm2 and 20 +/- 6 X 10(-6) ml/min, respectively; P less than 0.01] than in group 1 (7 +/- 6 sites/cm2 and 7 +/- 5 X 10(-6) ml/min), group 3 (9 +/- 5 sites/cm2 and 8 +/- 2 X 10(-6) ml/min), group 4 (11 +/- 7 sites/cm2 and 9 +/- 4 X 10(-6) ml/min), and group 5 (11 +/- 6 sites/cm2 and 7 +/- 1 x 10(-6) ml/min). The leaky sites appeared predominantly in postcapillary venules. There was a positive and significant correlation between the number of leaky sites and FITC-D clearance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ghrelin is suppressed by glucagon and does not mediate glucagon-related growth hormone release

BACKGROUND: Glucagon stimulation is routinely used as a provocative test to assess growth hormone (GH) sufficiency in pediatrics. Ghrelin also markedly stimulates GH secretion. Because glucagon stimulates the promoter of the ghrelin gene in vitro as well as ghrelin secretion by the perfused rat stomach, we sought to determine whether ghrelin mediates glucagon-induced GH secretion. METHODS: We compared ghrelin, GH, insulin and glucose responses following administration of 0.03 mg/kg intravenously (iv; max. 1 mg) and 0.1 mg/kg intramuscularly (im; max. 2 mg) of glucagon in two groups (n = 10-11/group) of GH-sufficient children. We also measured ghrelin before and 6 min after iv administration of 1 mg glucagon in 21 adult subjects. RESULTS: In children, glucagon caused a 26% decrease in ghrelin and a 72% increase in glucose concentrations that were independent of the dose or administration route of glucagon. In contrast, the insulin response was 2-3 times higher following administration of 0.1 mg/kg im compared to 0.03 mg/kg of glucagon iv. There was a significant correlation between the maximum decrease in ghrelin and increases in glucose (p = 0.03) but not in insulin. There was a significant correlation between ghrelin and GH area under the curve after controlling for the dose of glucagon (p = 0.03) but not for the maximum increase in glucose.In normal adults, glucagon administration caused a 7% decrease in ghrelin concentrations after 6 min (p = 0.0002). CONCLUSION: Ghrelin does not play a causal role in the GH response to pharmacological glucagon administration, which suppresses ghrelin levels starting a few minutes after injection.