Functional Characterization of Dipeptide Transport System in Human Jejunum

The present studies were performed to determine whether dipeptide absorption in human jejunum exhibits the characteristics of carrier-mediated transport. 15-cm jejunal segments from human volunteers were perfused with test solutions containing varying amounts of either glycylglycine, glycylleucine, glycine, leucine, glycylglycine with leucine or glycine, glycylglycine with glycylleucine, or glycylleucine with an equimolar mixture of free glycine and leucine. Jejunal absorption rates of both glycylglycine and glycylleucine followed the kinetics of a saturable process. The K(m) value in millimoles/liter of glycylglycine was significantly greater than the K(m) value of glycylleucine (43.3+/-2.6 vs. 26.8+/-5.9, P < 0.05); and the K(m) value of glycine was also significantly greater than the K(m) value of leucine (42.7+/-7.5 vs. 20.4+/-5.4, P < 0.05). While overlapping occurred among the K(m) values of free amino acids and dipeptides, the transport kinetics of dipeptides were characterized by higher V(max) values (in micromoles per minute per 15 centimeters) than those of free amino acids. For example, the V(max) values for glycylglycine and glycine were 837+/-62 and 590+/-56, respectively (P < 0.02). While jejunal absorption rates of glycylglycine were not significantly affected by free leucine or free glycine, they were competitively inhibited by glycylleucine. The jejunal absorption rate of glycylleucine was not significantly altered by an equimolar mixture of free glycine and leucine. The selective absorption of dipeptides was investigated by infusing three equimolar mixtures, each containing two different dipeptides. Among the three dipeptides examined, glycylglycine was the least absorbed. There was no significant difference between the absorption of glycylleucine and leucylglycine.The above studies suggest that absorption of both glycylglycine and glycylleucine is mediated by a carrier which is not shared with free neutral amino acids; and that both COOH- and NH(2)-terminal amino acids appear to be influential in imposing the affinity of a dipeptide for the absorption sites.     

Evidence for two different modes of tripeptide disappearance in human intestine. Uptake by peptide carrier systems and hydrolysis by peptide hydrolases.

The intestinal fate of two tripeptides (triglycine and trileucine), which differ markedly in solubility and molecular weight, have been investigated by jejunal perfusion in healthy human volunteers. Rates of glycine or leucine uptake from test solutions containing triglycine or trileucine were greater than from test solutions containing corresponding amounts of free glycine or free leucine, respectively. The rate of glycine uptake from a 100 mM triglycine solution was greater than that from a 150 mM diglycine solution. At each infused load of triglycine (e.g., 1,000 mumol/min) the rates (micromoles/minutes per 30 cm) of either triglycine disappearance (810 +/- 40) or glycine absorption (2,208 +/- 122) were markedly greater than the luminal accumulation rates of either diglycine (56 +/- 10) or free glycine (110 +/- 18). The luminal accumulation rate of free leucine during infusion of a 5 mM trileucine solution was over threefold greater than that of free glycine during the infusion of a 5 mM triglycine solution. Luminal fluid exhibited no hydrolytic activity against triglycine, but contained some activity against trileucine. Saturation of free amino acid carrier system with a large load of leucine did not affect glycine absorption rate from a triglycine test solution, but isoleucine markedly inhibited the uptake from a trileucine solution. When the carrier system for dipeptides was saturated with a large amount of glycylleucine, the disappearance rate of triglycine was considerably reduced while that of trileucine remained unaffected. After addition of glycylleucine to tripeptide solutions, there was a minimal increase in the luminal accumulation of diglycine, while dileucine accumulation was incresed by 62-fold.     

Transport mechanisms of bestatin in rabbit intestinal brush-border membranes: role of H+/dipeptide cotransport system

Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine], a potent inhibitor of aminopeptidase B and leucine aminopeptidase, enhances the immune response to activate the defense mechanism of the living organism and suppresses the growth and metastasis of cancer. Bestatin has been effectively used by p.o. administration, but the mechanisms of intestinal absorption remain to be solved. The present study was undertaken to examine whether bestatin, a dipeptide containing an unusual amino acid, is transported via dipeptide carriers in intestinal brush-border membranes, by using cephradine as a probe for the H+/dipeptide cotransport system. The initial uptake of cephradine in the presence or absence of an inward H+ gradient, driving force, was inhibited by bestatin and this inhibition occurred in a competitive manner (Ki = 0.47 mM). The uptake of cephradine was stimulated by the countertransport effect of bestatin, the definitive criterion for ascertaining a common transport system. These findings indicate that bestatin, as well as cephradine and other p.o. cephalosporins, can be transported via dipeptide carriers in intestinal brush-border membranes.     

Intestinal transport of dipeptides in man: relative importance of hydrolysis and intact absorption

A 30 cm segment of the duodenum, jejunum, or ileum of normal human volunteers was perfused, on separate occasions, with test solutions containing either glycylglycine, free glycine, glycylleucine, or equimolar amounts of free glycine and free leucine. Luminal fluid contained no hydrolytic activity against glycylglycine and minimal activity against glycylleucine. In each intestinal segment, amino acid absorption rates were significantly greater from the test solutions containing the same amount of amino acids in dipeptide than in free form(as high as 185% increase). Perfusion of each intestinal segment with a test solution containing the equimolar mixture of free glycine and free leucine always resulted in a greater leucine than glycine absorption rate. This preferential absorption of leucine, however, was either diminished (jejunum) or almost abolished (duodenum and ileum) when the glycylleucine solution instead of the equimolar mixture was presented to the intestinal mucosa. Among the three segments, the duodenum exhibited the least potential for the disappearance of dipeptides. The jejunal and ileal dipeptide disappearance rates were either similar for glycylleucine (94% vs. 92%) or slightly different for glycylglycine (92% vs. 79%). Despite lack of a remarkable difference in the disappearance rates, absorption rates of constituent amino acids were markedly greater in the jejunum than in the ileum. This reduced amino acid absorption was brought about by a greater accumulation of free amino acids in the lumen of the ileal segment (3 to 10-fold difference). Inhibition of free glycine absorption by leucine during the perfusion of the intestine with a test solution containing glycylglycine and leucine did not result in any greater concentration of free glycine in the lumen than when the glycylglycine test solution did not contain free leucine. Similarly, inhibition of free glycine and free leucine absorption by isoleucine was not accompanied by any remarkable alteration of absorption rates of the constituent amino acids of glycylleucine. The results of these studies suggest that: (a) dipeptide disappearance in the gut lumen is principally accomplished by intact absorption and not by hydrolysis; (b) intracellular hydrolysis of dipeptides is markedly greater in the ileum than in the jejunum, while dipeptide absorption rates are either similar or only slightly different in these two segments; (c) there is no appreciable hydrolysis of glycylglycine by the membrane-bound enzymes and only a small fraction of glycylleucine is hydrolyzed by these enzymes.     

The effect of amino acid infusion on leg protein turnover assessed by L-[15N]phenylalanine and L-[1-13C]leucine exchange

A stable isotope technique depending on the use of [15N]phenylalanine and [1-13C]leucine to assess exchange was utilized to measure the components of protein turnover of the human leg and the effects of amino acid infusion. Eight healthy subjects (28.5 +/- 2.5 years) were studied when post-absorptive in the basal state and again during infusion of a mixed amino acid solution (55 g l-1, 1.52 ml kg-1 h-1). During the basal period leucine oxidation by the leg was 4.4 +/- 2.0 nmol 100 g-1 min-1 and this increased threefold during amino acid infusion (13.6 +/- 3.1 nmol 100 g-1 min-1, mean +/- SEM, P = 0.003). Amino acid infusion abolished the net negative balance between incorporation of leucine into, and release from, protein (basal, -31.8 +/- 5.8; during infusion, +3.1 +/- 7.1 nmol 100 g-1 P = 0.001). Phenylalanine exchange showed a similar pattern (basal, -13.7 +/- 1.8; during infusion, -0.8 +/- 3.0 nmol 100 g-1 min-1, P = 0.003). Basal entry of leucine into leg protein (i.e. protein synthesis) was 70.0 +/- 10.8 nmol 100 g-1 min-1 and this increased during amino acid infusion to 87.3 +/- 14.1 nmol 100 g-1 min-1 (P = 0.11). Phenylalanine entry to protein also increased with amino acid infusion (29.1 +/- 4.5 vs. 38.3 +/- 5.8 nmol 100 g-1 min-1, P = 0.09). Release from protein of leucine (101.8 +/- 9.1 vs. 84.2 +/- 9.1 nmol 100 g-1 min-1, P = 0.21) and of phenylalanine (42.8 +/- 4.2 vs. 39.1 +/- 4.2 nmol 100 g-1 min-1, P = 0.50) was unchanged by amino acid infusion. The results suggest that, in the post-absorptive state in man, infusion of mixed amino acids, without additional energy substrates; reverses negative amino acid balance by a mechanism which includes stimulation of muscle protein synthesis but which does not alter protein breakdown. Interpretation of the results obtained concurrently on whole-body protein turnover suggests that the increase in muscle protein synthesis contributes substantially to the whole-body increase, but the fall in whole-body breakdown with exogenous amino acids is independent of changes in muscle.     

Transport and metabolism of 6-thioguanine and 6-mercaptopurine in mouse small intestine

1. The transport of 6-thioguanine and 6-mercaptopurine has been studied with isolated jejunal loops of mouse small intestine. H.p.l.c. was used to identify and quantify the thiopurines and their metabolites in the serosal secretions. 2. When the lumen of the intestinal loops contained either 6-thioguanine or 6-mercaptopurine at a concentration of 1 mmol/l, the concentration of unmetabolized drug in the serosal secretions reached a maximum of 0.13 +/- 0.02 mmol/l (mean +/- SEM). 3. Analysis of the serosal secretions from the perfusions with either of the drugs revealed the appearance of an unknown compound which had the characteristics of a thiopurine and the same time course of appearance as the unmetabolized drug. Thus 6-thioguanine and 6-mercaptopurine are significantly metabolized during absorption in mouse intestine. 4. The unknown compound was identified as 6-thiouric acid, and with 1 mmol/l 6-thioguanine or 6-mercaptopurine in the lumen the concentration of this metabolite in the serosal secretions rose to a maximum of 0.13 +/- 0.01 and 0.18 +/- 0.03 mmol/l, respectively. At luminal drug concentrations of 0.1 mmol/l, the metabolite accounted for approximately 90% of the serosal thiopurine. 5. After an initial lag period of 20 min, linear rates of appearance in the serosal secretions were obtained for both the unmetabolized drugs and 6-thiouric acid. 6. Addition of the xanthine oxidase inhibitor oxypurinol at a luminal concentration of 0.3 mmol/l prevented the formation of 6-thiouric acid from 6-thioguanine. However, the inhibitor reduced the rate of 6-thioguanine appearance in the serosal secretions by 50%. 7. The conversion of 6-mercaptopurine to 6-thiouric acid was prevented when allopurinol or oxypurinol were added to the lumen. At a luminal drug concentration of 1 mmol/l, allopurinol increased the rate at which 6-mercaptopurine appeared in the serosal secretions by 90% compared with an increase of only 50% with oxypurinol. 8. The transport of water and glucose by the mouse intestinal loops was unaffected by 6-thioguanine or the xanthine oxidase inhibitors. However, 6-mercaptopurine caused significant reductions in the rate of water transport (30%) and glucose transport (39%). These effects were observed at a luminal drug concentration of 0.1 mmol/l and there was no further increase at a drug concentration of 1 mmol/l.     

Interaction of amino acids with glycl-L-leucine hydrolysis and transport in monkey small intestine.

1. There are two saturable transport processes in the monkey small intestine for glycyl-L-leucine, one with Vmax. 1 mumol min-1 g-1 wet weight of tissue and an affinity constant (kt) of 5 mmol/l, and the other with Vmax 3.9 mumol min-1 g-1 wet weight of tissue and kt 33 mmol/l. 2. Glycyl-L-leucine uptake is inhibited by a wide variety of amino acids, although to a variable extent. The inhibition was shown to be competitive with leucine used as the representative amino acid. Phenylalanine, methionine, alanine and leucine are the most potent in their inhibitory action. 3. The effect of various amino acids on the hydrolysis of glycyl-L-leucine by particulate and cytosol fractions of monkey small intestine was studied. All the amino acids, except glycine, proline, alanine and glutamic acid, inhibit both the particulate and cytosol glycyl-L-leucine hydrolase activities. In general, the cytosol enzyme is more susceptible to amino acid inhibition than the particulate enzyme. 4. There is no correlation between the effects of amino acids on glycyl-L-leucine uptake and hydrolysis of glycyl-L-leucine by either particulate or cytosol fraction.     

Effects of 2-(3-methyl-cinnamyl-hydrazono)-propionate on fatty acid and glucose oxidation in the isolated rat diaphragm using 14C-labelled substrates. Hydrazonopropionic acids, a new class of hypoglycaemic substances, VIII

The influence of 2-(3-methyl-cinnamyl-hydrazono)-propionate on the utilization of various substrates in isolated rat hemidiaphragms was investigated in comparison with other hypoglycaemic compounds. The effect of 2-(3-methyl-cinnamyl-hydrazono)-propionate was concentration-dependent. At a concentration of 0.5 mmol/l 2-(3-methyl-cinnamyl-hydrazono)-propionate, glucose utilization increased from 0.276 +/- 0.043 mumol.g-1.l-1 to 0.894 +/- 0.303 mumol.g-1.l-1 (p less than 0.05). Pyruvate and lactate utilization were stimulated to a lesser extent, while acetate utilization remained nearly constant. At a concentration of 2 mmol/l 2-(3-methyl-cinnamyl-hydrazono)-propionate, the oxidation of palmitate decreased from 0.214 +/- 0.017 mumol.g-1.l-1 to 0.060 +/- 0.005 mumol.g-1.l-1, while the oxidation of octanoate was not decreased. These findings point to a stimulation of the glycolytic flux by inhibition of long-chain fatty acid oxidation.     

Mucosal peptide hydrolase and brush-border marker enzyme activities in three regions of the small intestine of rats with experimental uraemia

1. The activities of nine peptide hydrolases and three non-peptidase brush-border marker enzymes have been quantified in crude homogenates prepared from the proximal, mild and distal regions of small-intestinal mucosa for sham-operated (n = 9) and uraemic (n = 14) rats. Abnormalities in enzyme activities were observed in all regions studied in the uraemic group, although no reduction in food intake occurred. 2. The proximal region of the small intestine from uraemic rats showed a general fall in enzyme activities associated with the brush-border. This fall was combined with a decline in mucosal protein content. In contrast, the mid and distal regions showed increased activity against the dipeptide tyrosyl-glycine. 3. It is proposed that the fall in brush-border enzyme activities in the proximal small intestine of uraemic rats is a response to the increased water intake associated with this, and presumably other, rat models of uraemia. The increased enzyme activity against tyrosyl-glycine found in the mid and distal regions of the small intestine of uraemic rats may be caused by an increased small-intestinal transit rate, but could be an attempt to maximize tyrosine absorption in response to decreased plasma tyrosine levels. 4. This study casts doubt on specific activities being the most useful units of enzyme activity, when measured in crude homogenates prepared from the proximal small intestine of uraemic rats. It also demonstrates that enzyme activities measured at a single site in the small intestine of uraemic rats may not be representative of the enzymatic changes occurring in the small-intestinal mucosa as a whole.     

Effects of fenoldopam on feline intestinal microcirculation

Experiments were performed to evaluate the effects of the specific dopamine-1-receptor agonist, fenoldopam, on the feline intestinal microcirculation. Cranial mesenteric arterial pressure, cranial mesenteric vein pressure, cranial mesenteric vein blood flow, venous occlusion capillary pressure, pre- and postcapillary resistances, total mesenteric vascular resistance, lymph flow, lymph and plasma protein concentrations, the capillary osmotic reflection coefficient, and the capillary filtration coefficient were determined in an isolated autoperfused jejunal segment in anesthetized fasted cats during intra-arterial administration of saline or fenoldopam mesylate in saline. Fenoldopam significantly increased mean cranial mesenteric vein blood flow from 26.8 +/- 3.4 to 33.7 +/- 2.8 ml.min-1.100 g-1. This increase in blood flow was due primarily to a significant decrease in mean intestinal vascular resistance from 3.14 +/- 0.32 to 2.54 +/- 0.2 mm Hg.ml-1.min-1.100 g-1, since cranial mesenteric arterial pressure during fenoldopam infusion was not different from the value obtained during control studies. Mean capillary pressure during fenoldopam infusion (17.2 +/- 0.5 mm Hg) was significantly greater than mean capillary pressure during control studies (15.6 +/- 0.3 mm Hg). The mean lymph flow during fenoldopam infusion (0.186 +/- 0.083 g/dl) was significantly greater than the value obtained during saline infusion (0.08 +/- 0.009 g/dl). Fenoldopam infusion significantly increased the mean capillary filtration coefficient from 0.135 +/- 0.021 to 0.275 +/- 0.035 ml.min-1.100 g-1.mm Hg-1 without altering the capillary osmotic reflection coefficient. These results suggest that specific dopamine-1-receptor stimulation in the small intestine increases the perfused capillary density without altering capillary permeability.(ABSTRACT TRUNCATED AT 250 WORDS)     

H+ coupled active transport of bestatin via the dipeptide transport system in rabbit intestinal brush-border membranes.

Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine], a dipeptide containing an unusual amino acid, has been used clinically as an anticancer agent in p.o. dosage form. We examined the transport characteristics of [3H]bestatin by rabbit intestinal brush-border membrane vesicles. Bestatin uptake was stimulated by an inward H+ gradient (overshoot phenomenon) and by an interior-negative membrane potential. About half of the apparent bestatin uptake at 1 mM by brush-border membrane vesicles was estimated as binding to the membranes. The affinity constant for the bestatin transport was 0.52 mM. The uptake of bestatin by brush-border membrane vesicles was inhibited by p.o. cephalosporins and dipeptides, but not by amino acids. In vesicles preloaded with either bestatin, cephradine or glycylsarcosine, the uptake of [3H]bestatin was stimulated markedly (countertransport effect). These results indicate that bestatin is transported via the H+/dipeptide transport system in rabbit intestinal brush-border membranes.     

Effects of spermine on water absorption, polyethylene glycol 4000 permeability and collagenase activity in rat descending colon in vivo.

1. The effects of spermine in the concentration range 0-10 mmol/l on (a) the fluid absorption, (b) the polyethylene glycol permeability, (c) the release of collagenase activity activity into the lumen and (d) the histological appearance of rat descending colon were examined. 2. Spermine (5 mmol/l) decreased fluid absorption from 48.83 +/- 2.98 (n = 7) to 23.98 +/- 2.32 (n = 6) microliters h-1 cm-2 (P < 0.01); polyethylene glycol 4000 permeability was increased from 0.030 +/- 0.001 (n = 7) to 0.047 +/- 0.003 (n = 6) cm/h (P < 0.01) and luminal collagenase activity increased from a negligible control value to 250 +/- 39 (n = 6) units/ml (P < 0.001). Spermine also caused oedema formation within the mucosal interstitial fluid, without inducing an overt breakdown of the mucosa at the luminal surface. 3. Polyamine-free dialysed seminal plasma had no effect on polyethylene glycol 4000 permeability, although it still caused a significant decrease in colonic fluid absorption from 48.83 +/- 2.98 (n = 7) (control) to 31.41 +/- 2.08 (n = 5) microliters h-1 cm-2 (P < 0.01). 4. Low-molecular-mass heparin (600 units/ml) prevented the spermine (5 mmol/l)- and whole-semen-induced increase in colonic polyethylene glycol 4000 permeability and reduced the effect of semen on fluid absorption by 63% (P < 0.001) and that of spermine by 56% (P < 0.01). 5. The Zn2+ chelator and collagenase inhibitor o-phenanthroline reduced the effect of spermine on fluid absorption and polyethylene glycol 4000 permeability by 100% (P < 0.001) and on interstitial oedema formation. o-Phenanthroline also reduced the effects of whole semen on fluid absorption (by 70%, P < 0.01) and on polyethylene glycol 4000 permeability by 95%, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbohydrate fermentation in the human colon and its relation to acetate concentrations in venous blood.

There is now substantial evidence that some dietary polysaccharides, notably dietary fiber, escape absorption in the small bowel and are then broken down in the large intestine of man. The main end products of this colonic digestive process, which is anerobic, are short chain fatty acids (SCFA), and acetic, propionic, and butyric acids. Although these acids are known to be absorbed from the colon, their subsequent fate and significance is unknown. We have measured venous blood SCFA levels in healthy subjects after a 16-h fast, and then following oral doses of either 50 mmol SCFA, 5, 10, or 20 g doses of the fermentable carbohydrate lactulose, or 20 g of pectin. Fasting venous blood acetate was 53.8 +/- 4.4 mumol/liter (SEM) (n = 14). Fasting arterial blood acetate, taken simultaneously with venous blood in six subjects, was higher; 125.6 +/- 13.5 mumol/liter (arterial) vs. 61.1 +/- 6.9 mumol/liter (venous). Significant levels of propionate or butyrate were not detected in any blood samples. Following an oral dose of 50 mmol mixed SCFA, venous blood acetate reached a peak of 194.1 +/- 57.9 mumol/liter at 45 min and returned to fasting levels at 2 h. Blood acetate also rose in response to lactulose, peak levels occurring 2-4 h after the dose: 5 g, 98.6 +/- 23.1 mumol/liter; 10 g, 127.3 +/- 18.2 mumol/liter; and 20 g, 181.3 +/- 23.9 mumol/liter. Pectin fermentation was much slower, with blood acetate levels starting to rise after 6 h and remaining elevated at about twice fasting levels for the subsequent 18 h. However, areas under the blood acetate curves were closely related (r = 0.97; n = 5), whatever the source of acetate. These studies show that the large intestine makes an important contribution to blood acetate levels in man and that fermentation may influence metabolic processes well beyond the wall of this organ.     

Potentiation by piridoxilate of the synthesis of hippurate from benzoate in isolated rat hepatocytes. An approach to the determination of new pathways of nitrogen excretion in inborn errors of urea synthesis

The synthesis of hippurate from benzoate as compared to ureagenesis has been investigated in isolated rat hepatocytes. Half-maximal synthesis of hippurate was observed at 0.3 mmol/l benzoate. In the presence of 1 mmol/l benzoate, hippurate synthesis proceeded linearly with time at a rate of 40 +/- 10 mumol X h-1 X g-1 dry weight. This provided less than 10% of nitrogen epuration supported by concomitant urea synthesis (350 +/- 82 mumol X h-1 X g-1 dry weight). The incorporation of benzoate to hippurate was markedly limited by the availability of glycine. Half-maximal hippurate synthesis was observed at 2 mmol/l glycine. In the absence of glycine, piridoxilate, a glyoxylate derivative, markedly potentiated hippurate synthesis. Half-maximal stimulation was observed at 10 mmol/l piridoxilate. In the presence of 10 mmol/l piridoxilate, hippurate synthesis (420 +/- 35 mumol X h-1 X g-1 dry weight) provided more than 50% of nitrogen epuration supported by urea synthesis. It is concluded that supplementation with nitrogen-free analogues of glycine such as piridoxilate are required to potentiate hippurate synthesis in an attempt to replace ureagenesis as an alternative pathway of waste nitrogen excretion in inborn errors of urea synthesis.     

Skeletal muscle and whole body protein turnover in thyroid disease

The effects of disturbances of thyroid hormone secretion on leg and whole body amino acid and protein metabolism have been investigated in seven patients with untreated thyrotoxicosis and eight patients with untreated hypothyroidism; the results were compared to those obtained in 11 normal control subjects. After treatment, the patients were restudied. Arterio-venous exchanges of tyrosine and 3-methylhistidine across leg tissue in the post-absorptive state were used as indices of net protein balance and myofibrillar protein breakdown, respectively. Whole body protein turnover was measured using stable isotope labelling techniques with 1-[1-13C] leucine. Efflux of tyrosine from leg tissues was six-fold greater in patients with untreated thyrotoxicosis than in normal control subjects (-19.39 +/- 2.21 vs. -4.20 +/- 0.31 nmol 100 g-1 leg tissue min-1, P less than 0.005, mean +/- SEM), but 3-methyl-histidine efflux was not significantly different (-0.11 +/- 0.03 nmol 100 g-1 leg tissue min-1 vs. 0.14 +/- 0.02 nmol 100 g-1 leg tissue min-1). After treatment, when the thyrotoxic patients became euthyroid, tyrosine efflux was normalized (at -4.94 +/- 0.84 nmol 100 g-1 leg tissue min-1) and 3-methylhistidine efflux was unchanged. In hypothyroid patients, neither tyrosine nor 3-methylhistidine effluxes were significantly different from those in normal subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of eprosartan on catecholamines and peripheral haemodynamics in subjects with insulin-induced hypoglycaemia

ANG II (angiotensin II) facilitates catecholamine release from the adrenal medulla and neuronal NE (noradrenaline) release. Since animal experiments point to specific sympatho-inhibitory properties of the AT1 (ANG II type 1)-receptor blocker EPRO (eprosartan), the primary aim of this study was to clarify if EPRO inhibits sympathetic reactivity in humans as determined by the effect of EPRO on insulin-induced catecholamine release. Sixteen healthy male volunteers were randomized in a double-blind cross-over study to receive a single dose of EPRO (600 mg) compared with placebo, followed by insulin-induced hypoglycaemia [0.15 IU (international unit)/kg of body weight; intravenous bolus] on two study days 1 week apart. From baseline to the end of hypoglycaemia (170 min), the sympatho-adrenal reactivity was mapped by invasive continuous blood pressure monitoring and repeated measurements of FBF (forearm blood flow), arterial and venous concentrations of glucose, catecholamines [EPI (adrenaline) and NE (noradrenaline)], renin, ANG II and aldosterone. EPRO induced an 8-10-fold increase in plasma renin and ANG II concentrations compared with placebo. Plasma glucose decreased equally during placebo and EPRO from baseline 5.9 mmol/l to 1.9 mmol/l and 2.1 mmol/l respectively, inducing a 17-fold increase in arterial EPI concentration at peak. The AUC (area under the curve) during hypoglycaemia for arterial EPI concentrations was 314+/-48 nmol.min.l-1 in placebo compared with 254+/-26 nmol.min.l-1 following EPRO treatment (P=0.14). EPRO attenuated the corresponding AUC for the EPI-induced pulse pressure response (4670+/-219 mmHg.min in EPRO compared with 5004+/-266 mmHg.min in placebo; P=0.02). Moreover, EPRO caused a less pronounced increase in FBF compared with placebo (402+/-30 compared with 479+/-46 ml.100 g-1 of body weight; P=0.04). Musculocutaneous NE release was not affected by EPRO and the AUC for NE release was 51.69+/-15.5 pmol.min-1.100 g-1 of body weight in placebo compared with 39.35+/-18.2 pmol.min-1.100 g-1 of body weight after EPRO treatment (P=0.57). In conclusion, EPRO did not significantly inhibit sympathetic reactivity compared with placebo; however, it blunted the haemodynamic responses elicited by the sympatho-adrenal stimulation which only tended to be attenuated by this drug.     

Glucose absorption from maltotriose and glucose oligomers in the human jejunum

The jejunal absorption of glucose from (1-4)-linked glucose oligomers including maltotriose has been compared with that from free glucose and sucrose in normal subjects. A steady-state perfusion technique in vivo was used to study proximal jejunal assimilation of isotonic sugar-saline solutions isocaloric with 140 mmol/l glucose. Endogenous alpha-amylase was excluded from the test segment by proximal balloon occlusion. The glucose oligomer mixture consisted mainly of maltotriose, maltotetraose, maltopentaose and maltoheptaose. Glucose absorption was significantly faster from maltotriose alone and from the glucose oligomer mixture than from 140 mmol/l glucose controls, whereas glucose absorption from 70 mmol/l sucrose was similar to that from 70 mmol/l glucose plus 70 mmol/l fructose. Hydrolysis in vivo of maltotriose, the oligomer mixture and sucrose were similar, indicating that capture of glucose released by brush border sucrose hydrolysis was less efficient than that associated with (1-4)-linked oligomer hydrolysis. This suggests that the stoichiometric relationship of the active hydrolysis sites for sucrose to the glucose transport system is less advantageous than that of active sites for maltose hydrolysis. Hydrolysis of oligomers larger than maltohexaose may be rate limiting for glucose absorption in the absence of luminal amylase activity.     

H+ gradient-dependent and carrier-mediated transport of cefixime, a new cephalosporin antibiotic, across brush-border membrane vesicles from rat small intestine

The characteristics of the transport of cefixime, a new p.o. cephalosporin, antibiotic, were studied by using brush-border membrane vesicles from the rat small intestine. The initial rate of uptake of cefixime was not affected by the presence of an inward gradient of either Na+ or other monovalent cations. With an intravesicular pHi of 7.5, optimal cefixime uptake occurred at an extravesicular pHo of 5.0, with about 6-fold acceleration compared with that in the absence of an inward proton gradient (pHi = pHo = 7.5). A protonophore, carbonyl-cyanide-4-trifluoromethoxy-phenylhydrazone, abolished the stimulating effect of low pHo. In the presence of a sufficient inward proton gradient (pHi = 7.5, pHo = 5.0), cefixime uptake showed an overshoot phenomenon and apparent saturation kinetics expressed by the Michaelis-Menten equation with the maximum rate of 2.67 +/- 0.06 nmol/30 sec/mg of protein and a Michaelis constant of 0.83 +/- 0.04 mM. Cefixime uptake was inhibited competitively by glycyl-L-proline and stimulated by the countertransport effect of this dipeptide. The other peptides also inhibited cefixime uptake significantly. A valinomycin-induced inside-negative K+-diffusion potential had a dramatic reducing effect on the uptake of dianionic cefixime. All the data obtained in this study demonstrate that cefixime transport across the brush-border membrane vesicles is carrier-mediated, independent of Na+ and dependent on a H+ gradient via the peptide transport systems.     

Formation of prostaglandins and leukotrienes by human lung tissue in vitro after activation by the calcium ionophore A23187

The formation of metabolites of arachidonic acid by the cyclo-oxygenase and lipoxygenase pathways were determined in human lung tissue, obtained from surgery. In this measurement the chopped tissue was incubated with the calcium ionophore A23187. Formation of metabolites from [1-14C] arachidonic acid was also determined. The metabolites were extracted, separated by HPLC and identified by measurement of the absorption spectrum at 280 nm, radioactivity, biological activity and by radioimmunoassay. 6-keto-prostaglandin F1 alpha (6-ketoPGF1 alpha), the metabolite of prostacyclin, is the cyclo-oxygenase product present in the highest amount (400 +/- 49 ng g-1), followed by PGD2 (162 +/- 59 ng g-1) thromboxane B2 (102 +/- 32 ng g-1) PGE2 (104 +/- 46 ng g-1) and PGF2 alpha (58 +/- 26 ng g-1). The amounts of the lipoxygenase products are: leukotriene B4 (LTB4), 163 +/- 100 ng g-1; LTC4, 63 +/- 31 ng g-1 and LTE4 121 +/- 34 ng g-1. From [1-14C] arachidonic acid higher amounts of the cyclooxygenase than of the lipoxygenase products were formed, with the exception of PGE2. The effects of several of these substances on the contraction of human small airway smooth muscle were measured. The contractions, induced by equivalent amounts of LTC4 and a synthetic analogue of thromboxane T X A2 were approximately one hundred times those induced by PGD2, PGF2 alpha and histamine. These results suggest that thromboxane A2 and LTC4 are the most important arachidonic acid metabolites that induce bronchoconstriction in the human lung.     

Pharmacokinetics of novel dipeptide ester prodrugs of acyclovir after oral administration: intestinal absorption and liver metabolism

The amino acid prodrug of acyclovir (ACV), valacyclovir (VACV), is an effective antiherpetic drug. Systemic availability of ACV in humans is 3 to 5 times higher after oral administration of VACV. Enhanced bioavailability of VACV has been attributed to its carrier-mediated intestinal absorption via hPEPT1 peptide transporter followed by rapid and complete conversion to ACV. An earlier report suggested that the dipeptide ester prodrugs of ACV possess high affinity toward the intestinal oligopeptide transporter hPEPT1 and therefore seem to be promising candidates in the treatment of oral herpes virus infections. In the present study, we have examined the bioavailability of a series of dipeptide prodrugs of ACV after oral administration in Sprague-Dawley rats with cannulated jugular and portal veins. The area under plasma-concentration time curves expressed as minutes microgram milliliter(-1) for total concentration of VACV (208.4 +/- 41.2), and the dipeptide prodrugs Gly-Val-ACV (GVACV) (416.1 +/- 140.9), Val-Val-ACV (VVACV) (147.7 +/- 89.3), and Val-Tyr-ACV (VYACV) (180.7 +/- 81.2) were significantly higher than that of ACV (21.2 +/- 5.2) upon intestinal absorption. Interestingly, the bioavailability of ACV after administration of GVACV was approximately 2-fold higher than VACV. There was significant metabolism by hepatic first pass effect of the dipeptide prodrugs as evident by the higher levels of ACV obtained after systemic absorption compared with intestinal absorption of GVACV and VVACV. The dipeptide prodrugs of ACV exhibited higher systemic availability of regenerated ACV upon oral administration and thus seem to be promising drug candidates in treatment of genital herpes infections.