Carnitine disposition before and during valproate therapy in patients with epilepsy

Free and total carnitine and acylcarnitine in plasma and urine samples was measured in 22 epileptic patients before and after 15 and 45 days of valproate (VPA) therapy and in 16 healthy volunteers on a single occasion. Carnitine plasma concentration and renal excretion observed in epileptic patients before VPA therapy did not differ from control values. After VPA was started, free and total plasma concentration decreased significantly (p < 0.05) from 49 +/- 17 to 35 +/- 16 at 15 days and to 35 +/- 13 nmol/ml at 45 days of therapy (free carnitine) and from 60 +/- 18 to 50 +/- 18 at 15 days and to 55 +/- 14 nmol/ml at 45 days of therapy (total carnitine), whereas acylcarnitine increased significantly (p < 0.05) from 10 +/- 8 to 14 +/- 8 at 15 days and to 18 +/- 16 nmol/ml at 45 days of therapy. Free carnitine urinary excretion decreased significantly (p < 0.05) from 200 +/- 135 to 115 +/- 76 and 118 +/- 75 mumol/24 h, whereas acylcarnitine urinary excretion increased significantly (p < 0.05) from 78 +/- 56 to 154 +/- 98 and 155 +/- 89 mumol/24 h after VPA therapy was started. As a consequence, acylcarnitine renal clearance increased significantly (+30%, p < 0.05) whereas free carnitine renal clearance did not change during VPA therapy. No difference was detected between 15 and 45 days of therapy. No patients experienced symptoms of VPA toxicity. Our results suggest that VPA in patients increases both formation and renal clearance of acylcarnitine.     

Retinal cholinergic system: characterization of rat retinal acetyltransferases using specific inhibitors of choline- and carnitine-acetyltransferases

Choline acetyltransferase catalyzes the synthesis of acetylcholine from choline and acetylcoenzyme A (ACoA) in both nervous and non-nervous tissues. Carnitine acetyltransferase occurs in several tissues and transfers acetyl groups from ACoA to carnitine forming acetylcarnitine and exhibits weak choline acetyltransferase activity. Several haloacetylcholines and haloacetylcarnitines were synthesized to develop selective inhibitors of choline acetyltransferase and carnitine acetyltransferase. Acetylcholine is a transmitter for some presynaptic neurons and/or amacrine cells in retina. Selective inhibitors of choline acetyltransferase and carnitine acetyltransferase were used in the evaluation of choline acetyltransferase and carnitine acetyltransferase activities in the rat retina. Choline acetyltransferase and carnitine acetyltransferase activities were assayed by transferring of [14C]acetyl group from [14C]ACoA to choline or carnitine and estimating [14C]-acetylcholine or [14C]acetylcarnitine. This study gave the following results: (a) Bromoacetylcholine (BrACh) was a selective inhibitor of purified choline acetyltransferase (I50, 2.2 microM); (b) (R)-bromoacetylcarnitine [(R)-BrACa] was more potent for inhibiting purified carnitine acetyltransferase (I50, 4 microM) than purified choline acetyltransferase (I50, 46 microM); (c) Rat retinal sonicate gave choline acetyltransferase activity of 98 +/- 6 nmol of ACh formed/mg/10 min. When the carnitine acetyltransferase was completely inhibited by (R)-BrACa, the activity for choline acetyltransferase decreased to 47 +/- 1 nmol, and this decrease was possibly due to the formation of some [14C]acetylcholine by carnitine acetyltransferase. The net retinal choline acetyltransferase activity was 51 nmol acetylcholine/mg protein/10 min; (d) Rat retinal sonicate contained carnitine acetyltransferase activity of 102 +/- 7 nmol acetylcarnitine formed/mg protein/10 min. This was not altered by inhibition of choline acetyltransferase with BrACh. This means that choline acetyltransferase did not use carnitine as a substrate. Choline acetyltransferase and carnitine acetyltransferase activities did not change after dialysis of retinal sonicates at 4 degrees C for 24 hrs. These observations suggest that BrACh and (R)-BrACa are useful for assessing the correct values for choline acetyltransferase and carnitine acetyltransferase activities in retinal tissues.     

Active transport of butyrobetaine and carnitine into isolated liver cells

1. The liver cells lose the major part of their carnitine during the commonly used isolation procedure by the collagenase-perfusion method. 2. The cells take up carnitine and the carnitine precursor butyrobetaine when these substances are added to the medium. The carnitine content of isolated liver cells can increase to about 15 mM with no apparent harm to the cells. 3. The data indicate the existence of a common carrier in the plasma membrane which mediates the uphill transport of both carnitine and butyrobetaine. The carrier has a high affinity for butyrobetaine (Km=0.5 mM) and a lower one for carnitine (Km=5.6 mM). 4. The intracellular butyrobetaine is hydroxylated to carnitine with a rate of approximately 0.33 mumol-g wet weight-1-h-1 which is sufficient to cover the turn over of carnitine in the whole rat. Carnitine is effectively esterified in the liver cells to acetylcarnitine and long-chain acylcarnitines. 5. Both carnitine and acetylcarnitine are released from the cells. The release of both compounds is probably physiological since it was found that acetylcarnitine constitutes a similar fraction of the total acid soluble carnitine both in the blood and liver of the intact rat.     

Acute effects of cell isolation on InsP profiles in adult rat cardiomyocytes

The isolation and culture of adult rat cardiomyocytes was shown to cause major changes in the contents of [3H]-labeled inositol phosphates and inositol phospholipids. Undigested heart tissue contained high levels of [3H]Ins(1,4,5)P3 (5364+/-800 ct/min/g tissue, 80+/-12 ct/min/mg protein) and mass content averaged 13.8 nmol/g tissue or 208+/-36 pmol/mg protein (mean+/-S.E.M., n=4). After collagenase digestion, [3H]Ins(1,4,5)P3 was undetectable and the mass content of Ins(1,4,5)P3 had decreased to 0.8+/-0.2 pmol/mg protein (mean+/-S.E.M., n=4, P<0.01). [3H]Ins(1,4)P2 was reduced by 80% and [3H]PtdIns(4,5)P2 by 90%. These profiles remained essentially unchanged when the isolated cells were maintained in culture for up to 24 h, even though the inositol phosphate response remained sensitive to norepinephrine. Similar to findings in intact tissue, the inositol phosphate response to norepinephrine in these cells was inhibited by neither U-73122 (5 microM) nor by neomycin (5 mM). By 48 h in culture, the relative levels of [3H]Ins(1,4,5)P3 and [3H]Ins(1,4)P2 had increased in relation to the total inositol phosphate content and responses appeared to better reflect intact tissue. However, while retaining insensitivity to neomycin, cells at 48 h were fully sensitive to U-73122 (5 microM). These data demonstrate that altered inositol phosphate responses are observed in adult cardiomyocytes from the time of isolation and that while the profiles change over time in culture, a pattern similar to that in intact heart is not re-established.     

Carnitine measurements in liver, muscle tissue, and blood in normal subjects

We determined carnitine concentrations in blood and in liver and abdominal muscle biopsy specimens in 13 men and 16 women undergoing elective surgery (mostly gallbladder removal). The data suggest that the carnitine pools of plasma and erythrocytes are different. The erythrocytes show a higher acylcarnitine concentration than does plasma (P < 0.001). Several reference bases for values in tissues have been used--dry weight, noncollagen protein (NCP), and DNA--because these may be differently influenced by disease. In liver specimens, the quotient NCP (g)/DNA (g) was significantly higher in men, 54.4 +/- 6.3 (mean +/- SD), than in women, 47.7 +/- 7.0 (P < 0.01). Liver total carnitine content in relation to DNA was significantly higher in men than in women: 0.29 +/- 0.06 vs 0.22 +/- 0.08 mmol/g DNA (P < 0.01). Free carnitine content was significantly higher in men than in women independently of the reference base, e.g., 3.7 +/- 1.0 mumol/g NCP for men vs 2.9 +/- 1.0 for women (P < 0.05). No difference was found between the sexes in the abdominal muscle carnitine content, 20.6 +/- 6.7 mumol/g NCP for men vs 17.9 +/- 5.0 for women. Our study establishes control ranges, thereby providing an important basis for studies of patients with abnormal carnitine metabolism.     

Origins of the extracellular glutamate released during total metabolic blockade in the immature retina

Previous studies have shown that complete blockade of metabolism in embryonic chick retina causes a time-dependent increase in the release of glutamate into the extracellular space. The present study examined the cellular source of this glutamate, i.e., neuronal and/or glial. Pure cultures of retinal neurons or glia were labeled for 10 min at 37 degrees C with [3H]acetate. Retinal glia, but not retinal neurons, were found to selectively and preferentially metabolize acetate, thus producing 3H-labeled amino acids in the glial compartment. This finding provides direct evidence to substantiate findings from several other laboratories that have indirectly determined the preferential metabolism of acetate by glia by using mixed neuronal/glial populations. To study the cellular source of glutamate released during total metabolic blockade, whole retina were prelabeled with [3H]acetate plus [U-14C]glucose (to label the neuronal compartment). Total metabolic blockade was instituted with a combination of iodoacetate (IOA) plus KCN, and the release of glutamate into the medium was followed at 5, 15, and 30 min. During total energy blockade, net extracellular glutamate was not elevated at 5 min [0.17 +/- 0.02 vs. 0.12 +/- 0.01 microM for treated vs. control retina (means +/- SEM), respectively], but was increased significantly at 15 (1.2 +/- 0.26 microM) and 30 min (2.6 +/- 0.22 microM). Total [3H]glutamate in the medium during IOA/KCN treatment was unchanged at 5 min, but was increased 1.5- and threefold above basal levels at 15 and 30 min, respectively. During the time when extracellular glutamate increased, the specific activity of [3H]glutamate remained fairly constant, 731 +/- 134 and 517 +/- 82 dpm/nmol (means +/- SEM) at 15 and 30 min, respectively. In contrast, 14C-labeled glutamate in the medium did not increase during IOA/KCN treatment and paralleled basal levels. Thus, the specific activity of 14C-labeled extracellular glutamate decreased from 309 +/- 87 dpm/nmol at 15 min to 42 +/- 8 dpm/nmol at 30 min. Prior loading of the tissue with 0.5 mM trans-pyrrolidine-2,4-dicarboxylate (t-PDC), a glutamate transport inhibitor, blocked 57% of the glutamate released at 30 min of IOA/KCN exposure, suggesting that reversal of an Na+-dependent glutamate transporter was a key contributor to the appearance of extracellular glutamate during energy deprivation. The increase in extracellular [3H]glutamate, constancy of the specific activity of extracellular [3H]glutamate, decrease in the specific activity of extracellular [14C]glutamate, and attenuation of release by prior loading with t-PDC indicate that glial pools of glutamate released via reversal of the transporter contribute significantly to the rise in extracellular glutamate after metabolic inhibition in this preparation.     

The gene encoding the catalytic subunit C alpha of cAMP-dependent protein kinase (locus PRKACA) localizes to human chromosome region 19p13.1

OBJECTIVE: The importance of portal hypertensive gastropathy, as a potentially bleeding lesion in cirrhotics with portal hypertension, has recently been appreciated. Histologically, dilation of the mucosal and submucosal vessels of the stomach is noted in this entity. The possibility of nitric oxide acting as a mediator for this mucosal vascular dilation has not been explored. METHODS: We determined, in a group of 10 male cirrhotic patients with esophageal varices and endoscopic changes consistent with severe portal hypertensive gastropathy (Group A), the gastric mucosal nitric oxide synthase activity. This was determined by measuring the rate of conversion of [3H]-arginine to [3H]-citrulline. Serum levels of nitrates and nitrites, the end products of nitric oxide, were also measured. The results were compared with those of a group of 10 male controls with no liver disease (Group B). RESULTS: Gastric mucosal constitutive and inducible nitric oxide synthase levels were significantly higher in group A (125.4 +/- 4.3 and 259.7 +/- 5.5 pmol/mg protein/minute, respectively) than in group B (88 +/- 8.6 and 130.8 +/- 6.6 pmol/mg protein/minute, respectively) ( p < 0.002 and < 0.0001, respectively). Serum nitrate/nitrite levels were 30.1 +/- 3.2 nmol/ml in group A and 15.5 +/- 0.09 nmol/ml in group B (p < 0.001). CONCLUSIONS: We conclude that the significantly increased gastric mucosal nitric oxide synthase activity, in patients with portal hypertensive gastropathy, suggests an important role for nitric oxide in the pathogenesis of this mucosal lesion.     

O-ethyl 14C]phenacetin O-deethylase activity in human liver microsomes

The activity of human liver microsomal cytochrome P450 1A2 (CYP1A2) is readily estimated by following the O-deethylation of [O-ethyl 14C]phenacetin (PODase). The basis of the assay is the quantitative measurement of [14C]acetaldehyde, remaining in the supernatant of assay incubates, after extraction of unmetabolized [O-ethyl 14C]phenacetin with charcoal. In the presence of native human liver microsomes (K(m) = 54 +/- 27 microM; V(max) = 14 +/- 2.3 nmol/hr/mg; mean +/- SD; N = 3 different livers) and human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP1A2 (K(m) = 46 microM; V(max) = 55 nmol/hr/nmol CYP), PODase activity conformed to monophasic Michaelis-Menten kinetics. Furthermore, PODase activity in a panel of microsomes prepared from a series of human livers was significantly correlated (r = 0.91; p < 0.001; N = 11) with CYP1A2-selective 7-ethoxyresorufin O-deethylase activity, and was markedly inhibited (> or = 92%) by furafylline (FURA, IC50 = 0.4 microM) and 7,8-benzoflavone (ANF, IC50 = 0.1 microM), two well known CYP1A2 inhibitors. Inhibitors selective for other forms of CYP (e.g. CYP3A, CYP2C, CYP2D6, CYP2E1) elicited a marginal effect (< or = 17% inhibition) at relatively high concentrations (> or = 10.K(i)). It is concluded that the inhibition of human liver microsomal CYP1A2 activity can be readily determined by using a charcoal-based radiometric method employing [O-ethyl 14C]phenacetin as substrate.     

Correlation of nicotinic binding with neurochemical markers in Alzheimer's disease

The loss of neocortical synapses that occurs in Alzheimer's disease (AD) has been shown to correlate with cognitive decline. In addition, marked losses in the cholinergic system in AD, specifically choline acetyltransferase (ChAT) activity and high affinity presynaptic neuronal nicotinic cholinergic receptors (nAChRs), have also been described. We hypothesized that in AD, the loss of [3H]-ligand binding to nAChRs, which are largely presynaptic, would correlate with changes in two other presynaptic markers: synaptophysin (Syn), a measure of synaptic density, and ChAT activity. The midfrontal (MF) cortex of 36 autopsy confirmed (NIA and CERAD criteria) AD patients (mean death age +/- SD 80.1 +/- 8.4 years) who met NINDS-ADRDA criteria for a clinical diagnosis of probable or possible AD, and 11 nondemented controls (mean death age +/- SD 77.9 +/- 8.0) were examined. Synapse counts were quantified by a dotimmunobinding assay for Syn. ChAT activity was assessed by standard biochemical assays. Nicotinic cholinergic receptor binding was assayed using the high affinity nicotinic agonist [3H]-(+/-)-epibatidine ([3H]-EPI). The mean +/- SD Syn in AD (83.4 +/- 31.9 arbitrary units (AU)/mg protein) was significantly lower than controls (126.1 +/- 19.9, p = 0.0003; t-test). The mean ChAT activity in AD (139.0 +/- 75.6 nmol ACh/hr/100 mg protein) was significantly lower than controls (219.6 +/- 70.8, p = 0.004). The mean [3H]-EPI total binding in AD (6.2 +/- 2.8 fmol/mg protein) was significantly lower than controls (14.8 +/- 3.2; p < 0.0001). Syn correlated with [3H]-EPI binding in AD (r = 0.48, p = 0.006; Pearson) but ChAT did not (r = -0.20, p = 0.34). We conclude that loss of high affinity nAChR binding correlates with loss of synapses in AD. The lack of correlation between [3H]-EPI binding and ChAT activity suggests that the targeted receptor populations may not be located exclusively on cholinergic neurons.     

Effect of catechin on carbohydrate metabolism

The serum free carnitine levels of 33 children with recurrent pulmonary infection and 30 healthy children were measured and found to be 26.12 +/- 0.98 nmol/mL and that of the control group 38.98 +/- 0.79 nmol/mL on the average. The mean free carnitine level was statistically determined to be significantly lower when compared with that of the control group (P < 0.01). The results indicate that oral L-carnitine therapy is recommended for pediatric patients with recurrent pulmonary infection.     

Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles

Previous studies have shown that hypoglycemia may reduce counterregulatory responses to subsequent hypoglycemia in healthy subjects and in patients with diabetes. The effect of hypoglycemia on the hormonal response to a nonhypoglycemic stimulus is uncertain. To test the hypothesis that the cortisol response to corticotropin (ACTH) infusion is independent of antecedent hypoglycemia, 10 healthy subjects received a standard ACTH infusion (0.25 mg Cosyntropin [Organon, West Orange, NJ] intravenously over 240 minutes) at 8:00 AM on day 1 and day 3 and a hypoglycemic insulin clamp study (1 mU/kg/min) at 8:00 AM on day 2. During the hypoglycemic clamp, plasma glucose decreased from 5.0 mmol/L to 2.8 mmol/L for two periods of 120 minutes (mean glucose, 2.9 +/- 0.03 and 2.8 +/- 0.02 mmol/L, respectively) separated by a 60-minute interval of euglycemia (mean glucose, 4.7 +/- 0.01 mmol/L). Seven subjects also had paired control studies in random order during which a 330-minute euglycemic clamp (mean glucose, 5.0 +/- 0.11 mmol/L) instead of a hypoglycemic clamp was performed on day 2. Basal ACTH (4.6 +/- 0.7 v 2.6 +/- 0.4 pmol/L, P < .02) and basal cortisol (435 +/- 46 v 317 +/- 40 nmol/L, P < .02) both decreased from day 1 to day 3 following intervening hypoglycemia. In contrast, with intervening euglycemia, neither basal ACTH (5.9 +/- 1.5 v 4.5 +/- 1.0 pmol/L) nor basal cortisol (340 +/- 38 v 318 +/- 60 nmol/L) were reduced significantly on day 3 compared with day 1. Following interval hypoglycemia, the area under the curve (AUC) for the cortisol response to successive ACTH infusions was increased (4,734 +/- 428 nmol/L over 240 minutes [day 3] v 3,526 +/- 434 nmol/L over 240 minutes [day 1], P < .01). The maximum incremental cortisol response was also significantly increased (805 +/- 63 nmol/L (day 3) v 583 +/- 58 nmol/L (day 1), P < .05). In contrast, the AUC for the cortisol response to successive ACTH infusions with interval euglycemia (3,402 +/- 345 nmol/L over 240 minutes [day 3] v 3,709 +/- 391 nmol/L over 240 minutes [day 1] and the incremental cortisol response (702 +/- 62 nmol/L [day 3] v 592 +/- 85 nmol/L [day 1] were unchanged. Following exposure to intermittent hypoglycemia in healthy humans, fasting morning ACTH and cortisol levels are reduced and the incremental cortisol response to an infusion of ACTH is enhanced. The enhanced cortisol response to exogenous ACTH infusion after intervening hypoglycemia (but not intervening euglycemia) may reflect priming of the adrenal gland by endogenous ACTH produced during the hypoglycemia. These data suggest that adrenal function testing by exogenous ACTH administration is not impaired by prior exposure to hypoglycemia. Moreover, the reduced cortisol response to recurrent hypoglycemia in patients with well-controlled diabetes is not likely the result of impaired adrenal responsiveness.     

Low density lipoprotein enhances the thrombin-induced growth of vascular smooth muscle cells

OBJECTIVE: In the present study we investigated whether low density lipoprotein is able to enhance the growth promoting effects of thrombin in vascular smooth muscle cells. METHODS: DNA synthesis was examined by measurement of the [3H]thymidine incorporation into the cell DNA. Cell count was measured with a Neubauer cell box. Thrombin receptor mRNA was determined by Northern blotting. Ca2+ was measured by the fura 2-method. RESULTS: Thrombin (5 nmol/l), thrombin receptor activating protein (3 mumol/l) and low density lipoprotein (33 nmol/l) induce a 652 +/- 80%, 593 +/- 80% and a 316 +/- 60% increase in [3H]thymidine incorporation into DNA (mean +/- SD, n = 3), respectively. A coincubation of thrombin or thrombin receptor activating protein with low density lipoprotein led to a 1245 +/- 160% or 1200 +/- 40% increase of DNA synthesis (mean +/- SD, n = 3). Thus, coincubation of low density lipoprotein and thrombin causes a synergistic rather than an additive mitogenic effect on smooth muscle cells. Thrombin and low density lipoprotein induced a 22 +/- 8.4% and a 29% +/- 6% increase in cell number, respectively. Simultaneous treatment of vascular smooth muscle cells with thrombin and low density lipoprotein caused a 63 +/- 14% increase in cell number (mean +/- SD, n = 3). To further elucidate the underlying mechanism, we studied the effect of low density lipoprotein on the expression of thrombin receptor mRNA. Low density lipoprotein caused a 2.5-fold increase of thrombin receptor mRNA within 24 h, as assessed by Northern analysis. Preincubation of cells for 24 h with 33 nmol/l low density lipoprotein resulted in an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration from 538 +/- 54 to 923 +/- 75 nmol/l (mean +/- SD, n = 4). CONCLUSION: In summary, low density lipoprotein may enhance the mitogenic effect of thrombin probably by an up-regulation of thrombin receptor gene expression in vascular smooth muscle cells or by an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration.     

Importance of substrate changes in the decrease of hepatic glucose cycling during insulin infusion and declining glycemia in the depancreatized dog

We wished to determine whether the elevated glucose cycling (GC) between glucose and glucose-6-phosphate (G<-->G6P) in diabetes can be reversed with acute insulin treatment. In six insulin-deprived, anesthetized, depancreatized dogs, insulin was infused for 6-9 h at a starting dose of 45-150 pmol.kg-1.min-1 to normalize plasma glucose from 23.9 +/- 1.4 to 5.0 +/- 0.4 mmol/l and gradually decreased to and maintained at a basal rate (1.7 +/- 1.0 pmol.kg-1.min-1) during the last 3 h. GC, measured with [2-3H]- and [6-3H]glucose, fell markedly from 15.3 +/- 2.7 and normalized at 1.3 +/- 0.6 mumol.kg-1.min-1 (P < 0.001). This occurred because total hepatic glucose output fell much more (from 41.2 +/- 3.1 to 11.6 +/- 1.2) than did glucose production (from 25.9 +/- 1.9 to 10.3 +/- 1.0 mumol.kg-1.min-1) (both P < 0.01). Freeze-clamped liver biopsies were taken at timed intervals for measurements of hepatic enzymes and substrates. The elevated hepatic hexose-6-phosphate levels decreased with insulin infusion (151 +/- 24 vs. 71 +/- 13 nmol/g, P < 0.01). Maximal activities of glucose-6-phosphatase (G6Pase) (from 17.6 +/- 0.8 to 19.6 +/- 2.6 U/g) and glucokinase (from 1.1 +/- 0.2 to 1.0 +/- 0.2 U/g) did not change. Insulin infusion resulted in a threefold increase (P < 0.05) in the activity of glycogen synthase (active form), but had no effect on hepatic glycogen content.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of propionyl L-carnitine on skeletal muscle metabolism in renal failure

The effect of propionyl L-carnitine on skeletal muscle metabolism in chronic renal failure. Carnitine deficiency, resulting in defective oxidative ATP synthesis, has been implicated in the myopathy of chronic renal failure. Using 31P magnetic resonance spectroscopy we examined calf muscle metabolism in 10 dialysed patients before and after 8 weeks of propionyl L-carnitine (PLC) 2 g.p.o. daily. Resting phosphocreatine/ATP (4.41 +/- 0.20 [SEM]) decreased to normal control levels on PLC (3.98 +/- 0.14; controls 4.00 +/- 0.06). In contrast, there was no effect of PLC on aerobic and anaerobic metabolism of muscle during or following 2-10 min exercise. The maximal calculated oxidative capacity (Qmax) remained below normal (28 +/- 3 mM/min before and 24 +/- 3 mM/min after PLC; controls 49 +/- 3 mM/min). Qmax correlated positively with hemoglobin concentration ([Hb]) after PLC (p < 0.03). Oxidative capacity assessed by phosphocreatine recovery T significantly improved with PLC administration (0.93 +/- 0.1 to 0.74 +/- 0.08 min) in those patients (n = 6) with [Hb] > 10 g/dl. [Hb] was rate limiting to oxidative metabolism in recovery from exercise but only following treatment with PLC. Patients with anemia or those subjects who use relatively more non-oxidatively synthesized ATP during exercise, do not respond to PLC. Oxidative metabolism did not normalize on PLC suggesting that anemia and carnitine deficiency are not the only causes of mitochondrial dysfunction in renal failure.     

Neuropeptide Y in human hand veins: pharmacologic characterization and interaction with cyclic guanosine monophosphate-dependent venodilators in vivo

The dorsal hand vein compliance technique was used to study direct vascular effects of human neuropeptide Y in vivo. Human neuropeptide Y is an endogenous vasoconstrictor peptide that is costored with norepinephrine in sympathetic nerve endings and coreleased with the catecholamine under various physiologic and pathologic conditions. Compared with the alpha 1-adrenergic agonist phenylephrine (geometric mean dose-rate that produces the half-maximal response [ED50]: 1.05 nmol/min; maximum venoconstriction [Emax] +/- SEM, expressed as a percentage of baseline compliance: 91% +/- 3%), human neuropeptide Y was nine times more potent (geometric mean ED50: 0.122 nmol/min; p < 0.001) but markedly less efficacious (Emax: 58% +/- 4%; n = 12; p < 0.001). Venoconstrictor effects of human neuropeptide Y lasted several hours and were unchanged by simultaneous administration of alpha-adrenergic antagonists but were readily reversed by nitroglycerin or bradykinin. The high responsiveness of subcutaneous veins to human neuropeptide Y indicates that human neuropeptide Y may regulate venous compliance and filling of the venous subcutaneous capacitance bed in vivo.     

Autoantibodies to tissue transglutaminase as predictors of celiac disease

Our aim was to study the prognostic value of growth hormone (GH) -stimulated insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3) generation in patients with compensated [group 1 (N = 8) with a Child-Pugh (CP) score of 5-8] and decompensated postnecrotic liver cirrhosis [group 2 (N = 7) with a CP score of 9-12]. Serum levels of IGF-I, GH-binding protein (GHBP), and IGFBP-3 were measured before and 24 hr after a single subcutaneous injection of recombinant human GH (rhGH, 0.14 units/kg). Patients (mean age 56 years) were followed prospectively for three years. Six patients (40%) died during the follow-up period, of whom half had a CP score <9. Mean serum IGF-I levels 24 hr after rhGH injection (group 1 vs group 2, 17.4 +/- 6.8 vs 7.4 +/- 0.7 nmol/liter) predicted survival with 93% accuracy. Levels <10 nmol/liter portended a poor prognosis, with 15% survival at one year, whereas levels >10 nmol/liter had a 100% survival rate at one and two years, respectively. Baseline IGF-I (9.98 +/- 2.0 vs 6.38 +/- 0.8 nmol/liter), GHBP (9.2 +/- 3 vs 5.7 +/- 0.8%/50 microl), and IGFBP-3 serum levels at baseline (1.7 +/- 0.3 vs 0.86 +/- 0.2 mg/liter) and at 24 hr (2.04 +/- 0.38 vs 0.99 +/- 0.3 mg/liter) did not add to the predictive value of stimulated IGF-I levels at 24 hr and were less accurate in predicting the outcome in comparison to CP score (80%). We conclude that stimulated IGF-1 <10 nmol/liter may be a true predictor of a negative prognosis in patients with liver cirrhosis.     

Effects of vasodilators and perfusion pressure on coronary flow and simultaneous release of nitric oxide from guinea pig isolated hearts

OBJECTIVE: The aims were to validate the use of a direct reading NO electrode, to compare the effects of diverse acting drugs on altering coronary flow (CF) and NO release, and to examine the effects of altered perfusion pressure on flow-induced changes in NO concentration [NO] in the hemoglobin free effluent of guinea pig isolated hearts. METHODS: Hearts were isolated and perfused initially at a constant perfusion pressure (55 mmHg) with a modified Krebs-Ringer's solution equilibrated with 97% O2 and 3% CO2 at 37 degrees C. Heart rate, left ventricular pressure, CF, and effluent pH, pCO2, pO2, and NO generated current were monitored continuously on-line. Effluent was sampled for L-citrulline. Percent O2 extraction and O2 consumption were calculated. [NO] was quantitated with a sensitive amperometric sensor (sensitivity > or = 1 nmol/l approximately 3 pA) and a selective gas permeable membrane. RESULTS: The electrode was not sensitive to changes in solution pO2, flow, or pressure. The electrode was sensitive to pCO2 (-0.50 nmol/l/mmHg) and temperature (+24.5 nmol/l/degree C), so coronary effluent pCO2 was measured to compensate for a small decrease in pCO2 that occurred with an increase in coronary flow, and effluent temperature was rigidly controlled. Serotonin, bradykinin, and nitroprusside increased NO release along with CF, whereas nifedipine, butanedione monoxime, zaprinast, and bimakalim comparably increased CF but did not increase [NO] or NO release. Increases in CF (ml/g/min) and NO release (pmol/g/min), respectively, were 5.0 +/- 1 and 100 +/- 17 for 1 mumol/l serotonin, 7.5 +/- 1 and 148 +/- 18 for 100 nmol/l bradykinin, and 7.8 +/- 1 and 173 +/- 28 for 100 mumol/l nitroprusside. The increases in effluent NO by bradykinin were proportional to the increases in L-citrulline. Tetraethylammonium decreased CF, but did not change NO release, indomethacin changed neither CF nor NO release, and NG-nitro-L-arginine methyl ester (L-NAME) reduced CF by 2.6 +/- 1 ml/g/min and NO release by 25 +/- 8 pmol/g/min. An increase of CF of 8.0 +/- 0.3 ml/g/min, produced by increasing perfusion pressure from 25 to 90 mmHg, increased [NO] by 30 +/- 4 nmol/l; L-NAME but did not reduce the pressure-induced increase in CF, but reduced the increase in [NO] to 10 +/- 5 nmol/l. CONCLUSIONS: This study demonstrates in intact hearts real-time release of NO by several vasodilator drugs and by pressure-induced increases in flow (shear stress) and attenuation of these effects by L-NAME.     

Kynurenic acid distribution into brain and peripheral tissues of mice

Kynurenic acid (KYN), an antagonist of excitatory amino acid receptors, is a putative antidote against neuroexcitatory amino acid toxicity. We studied various doses (0.05-3.17 mmol/kg, i.p.) and the effects of probenecid coadministration (0.70 mmol/kg, i.p.) on tissue distribution of KYN in male and female Swiss-Webster mice. After injection of [3H]KYN, samples of brain, heart, liver, kidney, skeletal muscle, and gut were collected at selected times and assayed for KYN by liquid scintillation counting. The substance was absorbed rapidly and distributed into all tissues. Its content (nmol/g, mean +/- SE) at 60 min was 0.26 +/- 0.05, 1.80 +/- 0.05, and 40.4 +/- 8.1 in brain (for 0.05, 0.53, and 3.17 mmol/kg), 1.43 +/- 0.11, 14.3 +/- 3.7, and 212 +/- 32 in heart, 1.16 +/- 0.21, 10.6 +/- 2.6, and 254 +/- 21 in liver, and 7.41 +/- 2.65, 180 +/- 63, and 1899 +/- 254 in kidney. Net accumulation of KYN in brain was much lower than in other tissues. Probenecid increased KYN concentration in brain 2.5-fold. Peak brain:blood concentration ratio occurred between 60 and 180 min, was inversely associated with dose, and was not affected by probenecid. Although brain content was similar, female mice had an earlier peak brain:blood ratio (120 min) than males (180 min) for the 0.05 mmol/kg dose. Our results suggest the presence of a restricted transfer process for KYN with delayed egress from brain.     

No effect of kinins on DNA synthesis in LNCaP prostate cancer cells

1. Prostate has kininogenase activity and expresses members of the tissue kallikrein gene family. The present study examined the effect of exogenous and endogenous kinins on growth of LNCaP prostate adenocarcinoma cells. 2. Rate of DNA synthesis was measured by incorporation over 4 h of [3H]-thymidine into a TCA insoluble fraction of LNCaP cells that had been cultured for 24 h. 3. Increased [3H]-thymidine incorporation was seen in response to 10 nmol/L testosterone (+103 +/- 5 s.e.%), dihydrotestosterone (+113 +/- 14%) and R1881 (+64 +/- 10%) (P < or = 0.001; n = 4). 4. In contrast 0.05, 5 and 1000 nmol/L lysyl-bradykinin had no effect (15 +/- 4, 10 +/- 9 and 5 +/- 3 s.e.%, respectively; n = 7). Des-Arg9[Leu8]-bradykinin (a B1 receptor antagonist) and/or D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin (a B2 receptor antagonist), 1 nmol/L, and indomethacin, 5 mumol/L, also had little or no effect. 5. In conclusion, kallidin and endogenous kinins and prostaglandins have little or no effect on DNA synthesis and therefore on the growth of LNCaP cells in comparison to the two-fold stimulation produced by androgens.     

Hamster brown-adipose-tissue mitochondria. The role of fatty acids in the control of the proton conductance of the inner membrane

The specific ability of fatty acids to increase the proton conductance of the inner membrane of mitochondria from the liver and brown adipose tissue of cold-adapted hamsters was compared. The liver and brown-adipose-tissue mitochondria had their effective proton conductances increased by respectively 0.028 and 0.94 nmol H+- min-1. (mV of proton electrochemical gradient)-1 for each nmol of palmitate bound. No difference could be detected between the abilities of liver and brown-adipose-tissue mitochondria to bind fatty acids. Purine nucleotides did not displace farry acids from the brown-adipase-tissue mitochondria. The endogenous fatty acid content of hamster brown-adipose-tissue mitochondria prepared in the absence of album was found to be equivalent to 17 +/- 7 nmol of palmitate/mg protein. The fatty acid content was reduced to 1 nmol/mg after preincubation of the mitochondria with CoA, ATP and carnitine. No inert pool of fatty acids could be detected. The endogenous fatty acids of hamster liver mitochondria were less than 4 nmol of palmitate equivalent/mg protein. Some of the fatty acid associated with the brown-adipose-tissue mitochondria originates during preparation of the mitochondria. In the light of these results, the physiological role of the fatty acids in controlling the proton conductance of the brown-adipose-tissue mitochondrial inner membrane, and hence- non-shivering thermogenesis, is re-evaluated.