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 Number of Glycine Residues Which Limits Intact Absorption of Glycine Oligopeptides in Human Jejunum

Studies were performed to determine whether glycine peptides of four or more glycine residues can be transported by the peptide carrier system, previously shown to transport diglycine and triglycine. When human jejunum was perfused with tetraglycine solutions, the rate of tetraglycine disappearance increased linearly as the concentration was increased over the range of 12.5-50 mM, however, the rate was slow in comparison to diglycine and triglycine disappearance rates.     

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.     

Chloramphenicol accumulation by Haemophilus influenzae.

The mechanism of chloramphenicol transport into susceptible strains of Haemophilus influenzae cells has not been reported previously. We examined apparent uptake of chloramphenicol by bacterial cells by using high-pressure liquid chromatography to quantitate drug disappearance from liquid media. Cell-associated chloramphenicol concentration is 1,000-fold greater than the extracellular drug concentration. Under incubation conditions associated with chloramphenicol disappearance from media, cellular protein synthesis was inhibited; however, if accumulation was inhibited, protein synthesis occurred in the presence of the drug. Chloramphenicol uptake appeared saturable (Km = 0.96 mM, Vmax = 0.9 mumol/min per mg of protein) and energy dependent: disappearance from media was markedly decreased by 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, compounds which disrupt the proton motive force. Uptake occurred only in median which can support growth and was dependent upon temperature and pH. Drug accumulation was minimally affected by inhibitors of electron transport or by gentamicin and puromycin, both inhibitors of protein synthesis. The rate of disappearance was inhibited by SCH24893, a fluorinated chloramphenicol analog which also inhibits protein synthesis. We conclude that chloramphenicol accumulation by H. influenzae occurs by energy-dependent transport.     

Nitrogen absorption from isonitrogenous solutions of L-leucyl-L-leucine and L-leucine: a study in the isolated perfused rat small intestine.

1. We assessed the efficacy of nitrogen absorption from luminal L-leucine (1.2 and 12 mmol/l) and from isonitrogenous L-leucyl-L-leucine (0.6 and 6.0 mmol/l) in a preparation of vascularly and luminally perfused rat small intestine by measuring luminal leucyl-leucine disappearance and venous leucine appearance. 2. No intact dipeptide was found in the vascular perfusate. Leucine-nitrogen absorption, as judged by venous leucine appearance, was as efficient from free leucine (29 +/- 3 and 245 +/- 19 ng-atom min-1 g-1) as from leucyl-leucine (27 +/- 4 and 211 +/- 58 ng-atom min-1 g-1). It was not reduced in the presence of glycyl-L-proline or of the brush-border dipeptidase inhibitors alanine-beta-naphthylamide and cilastatin. 3. After one passage of the whole small intestine, only trace amounts of dipeptide, but large amounts of free leucine, were detected in the luminal effluent. Peptidase activity in the luminal effluent was demonstrated in 100,000 g supernatant and was inhibited by p-hydroxy-mercuribenzoate, but not by brush-border dipeptidase inhibitors. 4. We propose that nitrogen absorption from luminal leucyl-leucine may proceed predominantly via intraluminal peptide hydrolysis and subsequent transport of free leucine. Nevertheless, our findings and conclusions are at variance with previous observations and current opinion on intestinal handling of dipeptides, which may be due, in part, to the different methodological approach.     

Localization of the membrane defect in transepithelial transport of taurine by parallel studies in vivo and in vitro in hypertaurinuric mice.

We investigated the mechanism of taurinuria in three inbred strains of mice: A/J, a normal taurine excretor (taut+); and two hypertaurinuric (taut-) strains, C57BL/6J and PRO/Re. Plasma taurine is comparable in the three strains (approximately 0.5 mM), but taurinuria is 10-fold greater in taut- animals. Fractional reabsorption of taurine is 0.967 +/- 0.013 (mean +/- SD) in A/J); and 0.839 +/- 0.08 and 0.787 +/- 0.05 in C57BL/6J and PRO/Re, respectively. Taurine concentration in renal cortex intracellular fluid (free of urine contamination) is similar in the three strains. Taurine reabsorption is inhibited by beta-alanine, in taut+ and taut- strains. These in vivo findings reveal residual taurine transport activity in the taut- phenotype and no evidence for impaired efflux at basilar membranes as the cause of impaired taurine reabsorption. Cortex slices provide information about uptake of amino acids at the antiluminal membrane. Taurine behaves as an inert metabolite in mouse kidney cortex slices. Taurine uptake by slices is active and, at less than 1 mM, is greater than normal in taut- slices. Concentration-dependent uptake studies reveal more than one taurine carrier in taut+ and taut- strains. The apparent Km values for uptake below 1 mM are different in taut- and taut+ slices (approximately 0.2 mM and approximately 0.7 mM, respectively); the apparent Km values above 1 mM taurine are similar in taut+ and taut- slices. Efflux from slices in all strains in the same (0.0105-0.0113 mumol-min-1-g-1 wet wt), but taut- tissue retains about 10% more radioactivity over the period of efflux. beta-Alanine is actively metabolized in mouse kidney. Its uptake in the presence of blocked transamination, is greater; its intracellular oxidation is attenuated; and its exchange with intracellular taurine is diminished in taut- slices. These findings indicate impaired beta-amino acid permeation on a low-Km uptake system at the luminal membrane in the taut- phenotype. beta-Amino acids are not reclaimed efficiently either from the innermost luminal pool in cortex slices or from the ultrafiltrate in the tubule lumen in vivo. The former leads to high uptake ratios in vitro, the latter to high clearance rates in vivo. In vitro and in vivo data are thus concordant. This is the first time that a hereditary defect in amino acid transport has been assigned to a specific membrane surface in mammalian kidney.     

Metabolism of intravenously administered dipeptides in rats: effects on amino acid pools, glucose concentration and insulin and glucagon secretion.

1. Studies were performed to investigate the metabolic fate of dipeptides when administered intravenously in rats. Glycyl-leucine, glycylglycine or glycylsarcosine was injected into the jugular vein. The plasma disappearance rate after the peak plasma concentrations was most rapid for glycyl-leucine and least rapid for glycylsarcosine. 2. During urine collection for 40 min, trace amounts of glycyl-leucine and glycylglycine and 13% of the injected glycylsarcosine were excreted. 3. Neither glycylglycine nor glycyl-leucine was detected in the liver, muscle, intestinal mucosa or renal cortex, but concentrations of glycine or leucine, or both, in these tissues were increased after each injection. In contrast, glycylsarcosine was recovered in all these tissues with concentrations in the renal cortex being far greater than in any other tissue, but sarcosine was found only in the renal cortex and intestinal mucosa. 4. The changes in plasma concentrations of free amino acids, glucose and glucagon, and tissue concentrations of free amino acids, were similar after the intravenous administration of glycyl-leucine and an equimolar mixture of free glycine and leucine. However, the amount of insulin secreted during the 40 min after glycyl-leucine injection was 1-6 times that produced after the injection of the corresponding amino acid mixture. 5. Results show that, within the present experimental conditions, the intravenous administration of dipeptides is as effective as that of the corresponding free amino acids in enriching the tissue pools of amino acids. It is suggested that efficient hydrolysis by cellular enzymes prohibits accumulation of intact dipeptides in body tissues.     

Regulation of ammonia production by mouse proximal tubules perfused in vitro. Effect of luminal perfusion.

To investigate factors regulating ammonia (NH3) production by isolated defined proximal tubule segments, we examined the rates of total NH3 (NH3 + NH+4) production by individual proximal tubule segments perfused in vitro under a variety of perfusion conditions. Segments consisting of late convoluted and early straight portions of superficial proximal tubules were incubated at 37 degrees C in Krebs-Ringer bicarbonate (KRB) buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, pH 7.4. The rate of total ammonia production was calculated from the rate of accumulation of total NH3 in the bath. The total ammonia production rate by unperfused proximal segments was 6.0 +/- 0.2 (+/- SE) pmol/mm per minute, which was significantly lower than segments perfused at a flow rate of 22.7 +/- 3.4 nl/min with KRB buffer (21.5 +/- 1.4 pmol/mm per minute; P less than 0.001) or with KRB buffer containing 0.5 mM L-glutamine (31.9 +/- 2.5; P less than 0.001). The rate of NH3 production was higher in segments perfused with glutamine than in segments perfused without glutamine (P less than 0.01). The perfusion-associated stimulation of NH3 production was characterized further. Analysis of collected luminal fluid samples revealed that the luminal fluid total NH3 leaving the distal end of the perfused proximal segment accounted for 91% of the increment in NH3 production observed with perfusion. Increasing the perfusion flow rate from 3.7 +/- 0.1 to 22.7 +/- 3.4 nl/min by raising the perfusion pressure resulted in an increased rate of total NH3 production in the presence or absence of perfusate glutamine (mean rise in rate of total NH3 production was 14.9 +/- 3.7 pmol/mm per minute in segments perfused with glutamine and 7.8 +/- 0.9 in those perfused without glutamine). In addition, increasing the perfusion flow rate at a constant perfusion pressure increased the rate of luminal output of NH3. Total NH3 production was not affected by reducing perfusate sodium concentration to 25 mM and adding 1.0 mM amiloride to the perfusate, a condition that was shown to inhibit proximal tubule fluid reabsorption. These observations demonstrate that the rate of total NH3 production by the mouse proximal tubule is accelerated by perfusion of the lumen of the segment, by the presence of glutamine in the perfusate, and by increased perfusion flow rates. The increased rate of NH3 production with perfusion seems not to depend upon normal rates of sodium reabsorption. The mechanism underlying the stimulation of NH3 production by luminal flow is unknown and requires further study.     

Bicarbonate secretion in vivo by rat distal tubules during alkalosis induced by dietary chloride restriction and alkali loading.

To examine in vivo the separate effects on distal tubule JtCO2, of dietary chloride restriction, bicarbonate loading, and changes in luminal chloride concentration, we microperfused distal tubules at a physiologic flow rate (8 nl/min) with solutions containing either 45 or 0 mM chloride (after gluconate substitution). Rats were fed a diet containing zero, minimal, or normal amounts of chloride, while drinking either water or a solution of 0.15 M sodium bicarbonate. Neither extracellular fluid volume contraction nor negative chloride balance ensued. Analysis of covariance with repeated measures demonstrated that dietary chloride, drinking sodium bicarbonate, and perfusion with either 45 mM or zero chloride, each have separate and significant modulating effects on distal tubule bicarbonate secretion. During mild alkalemia, there is modest bicarbonate secretion which is significantly different from zero (-9.9 +/- 3.2 pmol.min-1.mm-1, P less than 0.01), and which is suppressed after perfusion with zero chloride. In contrast, during more pronounced metabolic alkalosis after supplemental bicarbonate drinking, the bicarbonate secretory flux is brisk (-26 +/- 3 pmol.min-1.mm-1) and significantly different from zero and persists (-11 +/- 3 pmol.min-1.mm-1) even during perfusion with zero luminal chloride. Accordingly, in this two-day model of alkalosis induced by dietary chloride restriction, there is regulatory secretion of bicarbonate by distal tubules in vivo which is modulated by luminal chloride concentration.     

Cl- secretion induced by bile salts. A study of the mechanism of action based on a cultured colonic epithelial cell line.

When applied to the basolateral (serosal) side of the T84 colonic epithelial monolayer, taurodeoxycholate caused net Cl- secretion in a dose-dependent manner with a threshold effect observed at 0.2 mM. In contrast, when applied to the apical (luminal) surface, concentrations of taurodeoxycholate below 1 mM had little or no effect. Only when the concentration of taurodeoxycholate present on the apical side was greater than or equal to 1 mM did apical addition results in an electrolyte transport effect. This apical effect on electrolyte transport was associated with an abrupt increase in the permeability of the monolayer. Cyclic AMP and cyclic GMP in the T84 monolayers were not increased by the bile salt, but in the presence of extracellular Ca2+, free cytosolic Ca2+ increased with a graded dose effect and time course that corresponded approximately to the changes in short circuit current (Isc). The results suggest that luminal bile salts at a relatively high concentration (greater than or equal to 1 mM) increase tight junction permeability. Once tight junction permeability increases, luminal bile salts could reach the basolateral membrane of the epithelial cells where they act to increase free cytosolic Ca2+ from extracellular sources. The resulting increases in free cytosolic Ca2+, rather than in cyclic nucleotides, appear to be involved in transcellular Cl- secretion.     

Jejunal Absorption and Secretion of Calcium in Patients with Chronic Renal Disease on Hemodialysis

10 patients with chronic renal disease on hemodialysis and 8 normals were studied by constant jejunal perfusion of calcium gluconate solutions, using polyethylene glycol as a nonabsorbable marker. Results in normals indicated that calcium absorption from 1 and 5 mM calcium solutions is mainly active. Absorption from 5, 15, and 20 mM solutions was a linear function of luminal calcium concentration, suggesting that the active transport carrier is saturated when luminal calcium concentration is greater than 5 mM and indicating that the increment in absorption at higher luminal concentrations is mainly the result of passive absorption. With 1 mM calcium, normals absorbed calcium against a concentration gradient, whereas the patients secreted calcium. Absorption in the patients was much less than normal with 5, 15, and 20 mM luminal calcium concentrations; however, the slope of this linear (passive) portion of the curve was normal. Unidirectional calcium fluxes were measured with calcium-47. Flux out of the lumen was depressed 2.5-fold in the patients, but flux into the lumen was normal. Xylose, urea, and tritiated water were absorbed normally, indicating no generalized abnormality of jejunal transport in these patients. Endogenous calcium secretion, estimated by the amount of calcium added to a calcium-free solution, was normal in the dialysis patients. These results indicate that active calcium absorption is markedly depressed in patients with chronic renal disease who are receiving hemodialysis therapy. On the other hand, passive calcium movement and endogenous calcium secretions are normal.     

Direct evidence for the absence of active Na+ reabsorption in hamster ascending thin limb of Henle's loop.

The mechanisms of Na+ transport across cell membranes were investigated in the in vitro microperfused hamster ascending thin limb (ATL) of Henle's loop using a fluorescent Na+ indicator sodium-binding benzofuran isophthalate. The intracellular Na+ concentration ([Na+]i) of the ATL cells was 17.1 +/- 1.7 mM (n = 22) when the ATL was microperfused in vitro with Hepes-buffered solution containing 204 mM Na+. Elimination of metabolites such as glucose and alanine from the basolateral solution increased [Na+]i. Applying either 5 mM cyanide or 5 mM iodoacetic acid to the bath also increased [Na+]i. The elimination of K+ and the addition of 10(-4) M ouabain in the bath increased [Na+]i by 25.0 +/- 5.0 mM (n = 5) in 3 min and by 10.7 +/- 2.4 mM (n = 4), respectively. The elimination of luminal and basolateral Na+ resulted in a decrease in [Na+]i, indicating Na+ permeability of both the luminal and basolateral cell membranes. The luminal Na+ permeability was not affected by furosemide. The presence of luminal Na+ permeability and the basolateral Na+/K+ ATPase suggests the presence of net active reabsorption of Na+, which is not a physiologically important amount, in our estimation.     

Block of 45Ca uptake into synaptosomes by methylmercury: Ca++- and Na+-dependence

Block of Ca++ influx into isolated nerve terminals by the neurotoxicant methylmercury (MeHg) was studied for its dependence on extracellular Ca++ and Na+. Depolarization-independent entry of 45Ca++ was determined in rat forebrain synaptosomes incubated in 5 mM K+ solution. 45Ca++ uptake was similarly measured after 1 ("fast" phase) or 10 sec ("total") of elevated K+ (41.25 mM)-induced depolarization or after 10 sec of elevated K+-induced depolarization after synaptosomes had been predepolarized for 10 sec in Ca++- and MeHg-free solutions ("slow" phase). In 5 mM K+ solutions, MeHg concentrations of 125 microM and greater significantly reduced synaptosomal 45Ca++ uptake measured during 1 or 10 sec of incubation. In K+-depolarized synaptosomes, the estimated IC50 for block of total, fast and slow 45Ca++ uptake by MeHg is 75 microM; 250 microM MeHg reduced uptake by approximately 90%. The reversibility of block by extracellular Ca++ was tested by increasing the extracellular Ca++ concentration from 0.01 to 1.15 mM. When compared to control, 50 microM MeHg reduced total uptake of 45Ca++ by greater than or equal to 70% and reduced fast uptake by 20 to 60% at all concentrations of extracellular Ca++ tested. At Ca++ concentrations of 0.01 to 0.15 mM, MeHg (50 microM) reduced slow uptake by 75 to 90%, but did not affect slow uptake at higher Ca++ concentrations (greater than or equal to 0.30 mM). When the dependence of block of 45Ca++ uptake on extracellular Na+ was tested, equivalent levels of inhibition were caused by MeHg (25 microM) for fast uptake by synaptosomes in Na+-containing and Na+-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal bicarbonate reabsorption in the rat. III. Distal tubule perfusion study of load dependence and bicarbonate permeability.

Using continuous microperfusion techniques, we studied the load dependence of bicarbonate reabsorption along cortical distal tubules of the rat kidney and their bicarbonate permeability. Net bicarbonate transport was evaluated from changes in tracer inulin concentrations and total CO2 measurements by microcalorimetry. Bicarbonate permeability was estimated from the flux of total CO2 along known electrochemical gradients into bicarbonate-and chloride-free perfusion solution containing 10(-4) M acetazolamide. Transepithelial potential differences were measured with conventional glass microelectrodes. Significant net bicarbonate reabsorption occurred at luminal bicarbonate levels from 5 to 25 mM, and at perfusion rates from 5 to 30 nl/min. Bicarbonate reabsorption increased in a load-dependent manner, both during increments in luminal bicarbonate concentration or perfusion rate, reaching saturation at a load of 250 pmol/min with a maximal reabsorption rate of approximately 75 pmol/min.mm. Rate of bicarbonate reabsorption was flow dependent at luminal concentrations of 10 but not at 25 mM. During chronic metabolic alkalosis, maximal rates of reabsorption were significantly reduced to 33 pmol/min.mm. The bicarbonate permeability was 2.32 +/- 0.13 x 10(-5) cm/s in control rats, and 2.65 +/- 0.26 x 10(-5) cm/s in volume-expanded rats. Our data indicate that at physiological bicarbonate concentrations in the distal tubule passive bicarbonate fluxes account for only 16-21% of net fluxes. At high luminal bicarbonate concentrations, passive bicarbonate reabsorption contributes moderately to net reabsorption of this anion.     

Lead accumulation by rat renal brush border membrane vesicles

Pb++ accumulation by rat renal cortical brush border membrane vesicles was evaluated by in vitro incubation with rapid filtration technique. Pb++ uptake was time- and concentration-dependent, with apparent saturation of binding sites at 100 to 200 microM (5 sec initial rate experiments). Equilibrium binding studies (60-min incubation) showed that the ratio of bound Pb++ to free Pb++ was constant at 1.25 +/- 0.07 between 0.01 to 10 microM Pb, with decreasing bound to free ratios at higher concentrations. Osmotic experiments showed that Pb++ uptake was due primarily to membrane binding rather than intravesicular accumulation. Electrochemical gradients of NaCl, KCl or protons did not increase vesicle uptake of Pb++. Incubation of vesicles with a number of amino acids did not stimulate Pb++ uptake although two (cysteine and glutathione) and the chelators EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) completely blocked this process. Competition studies with a number of other metals (at 10 microM and 1 mM) showed that only Sn++ or Sn +, La , Fe++ or Fe and Cu++ produced significant reductions in Pb++ uptake whereas Mg++, Ca++, Zn++, Cd++ and Hg++ were without effect on this process. Release of 203Pb from preloaded vesicles was accelerated in the presence of either cysteine or Sn +. Prior in vivo exposure to Pb (3 mg of Pb/kg i.v.) reduced Pb uptake to 70% of that of vesicles prepared from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between several rare earth ions and calcium ion in vascular smooth muscle.

The effects of lutetium (Lu+++), europium (Eu+++) and neodymium (Nd+++) on 45Ca distribution, 45Ca movements and contractions were examined in rabbit aortic smooth muscle. Tension responses to norepinephrine were markedly inhibited by all three rare earth ions; K+-induced responses and those to histamine were partially blocked. Addition of 1.5 mM Lu+++, Eu+++ or Nd+++ to the bathing solution reduced 45Ca uptake (Nd+++ greater than Eu+++ greater than greater than Lu+++), produced a maintained increase in 45Ca efflux from strips of media-intimal layer washed out in a 0-Ca++ solution (Nd+++ greater than Eu+++ greater than Lu+++), and, when present during 45Ca incubation (before 45Ca washout), altered the release of 45Ca elicited by addition of 1.5 mM Ca++ during the 45Ca washout (inhibition by Nd+++ greater than Eu+++ greater than Lu+++). The 3-fold greater uptake of 45Ca in a 0-Ca++ and 1.5 mM Lu+++ solution than in an equivalent Sr++ solution results from a decreased effect of Lu+++ upon the slower 45Ca washout component. The actions of Lu+++ were quantitatively similar to those of low La+++ concentrations (0.05-0.10 mM), whereas those of Nd+++ and Eu+++ resembled effects obtained with equimolar concentrations of La+++. Thus, the three rare earth ions produce qualitatively similar alterations in 45Ca uptake and superficial binding. The difference observed can be attributed to variations in their affinity for superficial Ca++ binding sites and in their ability to block Ca++ uptake.     

Comparison of the effects of recombinant human insulin-like growth factor-I and insulin on glucose and leucine kinetics in humans.

To compare the metabolic effects of elevated plasma concentrations of IGF-I and insulin, overnight-fasted normal subjects were studied twice, once receiving IGF-I and once insulin at doses that resulted in identical increases in glucose uptake during 8-h euglycemic clamping. Recombinant human IGF-I or insulin were infused in one group at high doses (30 micrograms/kg per h IGF-I or 0.23 nmol/kg per h insulin) and in another group at low doses (5 micrograms/kg per h IGF-I or 0.04 nmol/kg per h insulin). Glucose rate of disappearance (measured by [6,6-D2]-glucose infusions) increased from baseline by 239 +/- 16% during high dose IGF-I vs 197 +/- 18% during insulin (P = 0.021 vs IGF-I). Hepatic glucose production decreased by 37 +/- 6% during high dose IGF-I vs 89 +/- 13% during insulin (P = 0.0028 vs IGF-I). IGF-I suppressed whole body leucine flux ([1-13C]-leucine infusion technique) more than insulin (42 +/- 4 vs 32 +/- 3% during high doses, P = 0.0082). Leucine oxidation rate decreased during high dose IGF-I more than during insulin (55 +/- 4 vs 32 +/- 6%, P = 0.0001). The decreases of plasma concentrations of free fatty acids, acetoacetate, and beta-hydroxybutyrate after 8 h of IGF-I and insulin administration were similar. Plasma C-peptide levels decreased by 57 +/- 4% during high doses of IGF-I vs 36 +/- 6% during insulin (P = 0.005 vs IGF-I). The present data demonstrate that, compared to insulin, an acute increase in plasma IGF-I levels results in preferential enhancement of peripheral glucose utilization, diminished suppression of hepatic glucose production, augmented decrease of whole body protein breakdown (leucine flux), and of irreversible leucine catabolism but in similar antilipolytic effects. The data suggest that insulin-like effects of IGF-I in humans are mediated in part via IGF-I receptors and in part via insulin receptors.     

Effects of Anion-Transport Inhibitors on NaCl Reabsorption in the Rat Superficial Proximal Convoluted Tubule

The effects of anion-transport inhibitors on volume reabsorption, and total CO(2) concentrations were examined by in vivo microperfusion of superficial proximal convoluted tubules of rats. The luminal perfusion solution was a high-chloride, low-bicarbonate solution like that in the in vivo late proximal tubule. The anion-transport inhibitors were only added to the luminal perfusion solutions.In tubules perfused with the control high-chloride solution, the rate of volume reabsorption (J(v)) was 2.3+/-0.2 nl/mm.min (n = 18), and the collected total CO(2) concentration was 4.0+/-0.3 mM. Furosemide (3 mM) caused a marked reduction in volume reabsorption to 0.8+/-0.3 nl/mm.min (n = 20) and only a slight increase in the total CO(2) concentration of collected samples of perfusate (7.8+/-0.5 mM). 0.8 mM acetazolamide caused a more pronounced rise in the collected total CO(2) concentrations to 10.7+/-0.5 mM but only a slight fall in J(v) to 1.7+/-0.3 nl/mm.min (n = 19). Hence, we inferred that inhibition of carbonic anhydrase only partially accounted for the inhibition of J(v) by furosemide. 4-acetamido-4'-iso-thiocyanato-stilbene-2,2'-disulphonic acid (0.1 mM), a well-characterized inhibitor of erythrocyte anion exchange mechanisms, also reduced J(v) to 1.6+/-0.3 nl/mm.min (n = 15) without changing the total CO(2) concentrations of the collected perfusates (3.6+/-0.4 mM). The effect of 4-acetamido-4'-iso-thiocyanato-stilbene-2,2'-disulphonic acid on volume reabsorption could not be explained by carbonic anhydrase inhibition because there was no increase in the total CO(2) concentration of the collected fluids. Furosemide did not significantly inhibit the rate of tracer glucose efflux out of the tubules, which suggests that the effect of furosemide on volume reabsorption was not a result of some nonspecific depression of active sodium transport. These results are discussed with respect to the possible effects of anion-transport inhibitors on the paracellular shunt pathway, active sodium reabsorption, and neutral sodium chloride transport.     

Sodium chloride, urea, and water transport in the thin ascending limb of Henle. Generation of osmotic gradients by passive diffusion of solutes

Studies were designed to examine whether the thin ascending limb of Henle (tALH) decreases its luminal solute concentration by an active or a passive transport process. In all experiments isolated segments of rabbit tALH were perfused in vitro. When tubules were perfused with solutions identical to the bath, active transport of NaCl was excluded by the following: (a) osmolality of the collected fluid remained unchanged and the same as the bath. (b) net water reabsorption could not be demonstrated, and (c) transtubular potential difference was zero. Isotopic permeability coefficients (x 10(-5) cm s-1) were calculated from the disappearance rate of the respective isotope added to the perfusate. These values indicate that tALH is moderately permeable to [14C]urea (6.97 +/- 1.95) while having a higher permeability to 22Na (25.5 +/- 1.8) and [not readable: see text]Cl (117 +/- 9.1) than any other segment similarly studied. The influx (bath-to-lumen) isotopic permeabilities were not statistically different from the above efflux permeabilities. Osmotic water permeability was immeasurably small. When tALH were perfused with a 600 mosmol/liter solution predominantly of NaCl against a 600 mosmol/liter bath in which 50% of osmolality was NaCl and 50% urea (to simulate in vivo papillary interstitium), the collected fluid osmolality was decreased significantly below that of the bath (300 mosmol/liter/mm of tubule). The decrease in osmolality was due to greater efflux of NaCl as compared to influx of urea. We conclude that active transport of salt by the tALH was not detected by the experimental protocol of the current studies, and that the unique membrane characteristics of tALH allows for generation of osmotic gradients (lumen less concentrated than adjacent surroundings) on purely passive mechanisms when perfused with isosmolal salt solutions in a bath with appropriate salt and urea concentrations. These findings are consistent with the passive counter-current model previously proposed from this laboratory.     

Differential effects of histamine H2 receptor antagonists on amantadine uptake in the rat renal cortical slice, isolated proximal tubule and distal tubule

The interaction between amantadine and two histamine receptor antagonists was examined in the rat kidney. Amantadine (10 microM, 30 sec) was actively accumulated by cortical slices (slice/medium ratio = 0.4 +/- 0.3 [3.3 +/- 0.3 at 4 min], mean +/- S.E.M.), isolated proximal tubules (tubule/medium ratio = 35 +/- 1) and distal tubules (tubule/medium ratio = 19 +/- 2). In cortical slices, low cimetidine concentrations facilitated amantadine accumulation, whereas higher concentrations produced inhibition. Uptake in proximal tubules was enhanced by cimetidine and reached a maximum at approximately 100 microM. Cimetidine (20 microM) decreased the apparent Km (88 +/- 5 to 55 +/- 3 microM, P less than .005) without altering Vmax (6.8 +/- 0.5 to 5.8 +/- 0.6 nmol/mg/min). Conversely, cimetidine did not enhance uptake in distal tubules but elicited competitive inhibition at concentrations greater than 1 mM. Although this may partially delineate the differences observed between the cortical slice and proximal tubule data, such a discrepancy may also implicate additional sites of interaction in other segments of the cortical nephron and/or cimetidine inhibition of the relatively more significant luminal amantadine efflux in the proximal tubules. Ranitidine did not enhance amantadine accumulation but produced inhibition at high concentrations. In proximal and distal tubule preparations, ranitidine (10 mM) increased Km from 86 +/- 7 to 121 +/- 8 and 95 +/- 5 to 160 +/- 10 microM, respectively (P less than .05), whereas Vmax was not changed (8.9 +/- 0.7 to 7.9 +/- 0.8 and 4.3 +/- 0.1 to 3.8 +/- 0.2 nmol/mg/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)