Peter I. Karl

Characteristics of Histidine Uptake by Human Placental Microvillous Membrane Vesicles

ABSTRACT: L-histidine is an essential amino acid. Its fetal-to-maternal blood concentration ratio is high, suggesting active placental transport. In this study, human placental microvillous membrane vesicles were used to characterize L-histidine transport, heretofore not evaluated in human tissue. L-Histidine uptake by microvillous membrane vesicles was stimulated by an inward sodium gradient, leading to an “overshoot,” followed by apparent equilibration. Linear uptake at 22° C was limited to the 1st min. The initial sodium-dependent uptake rate was proportional to the sodium concentration in the medium. The sodium-dependent uptake was markedly diminished or lost when potassium, cesium, or choline was substituted for sodium but not lithium. Replacement of chloride with sulfate or gluconate had little effect. Sodium-stimulated Lhistidine uptake was further stimulated by an outward potassium gradient (inside-negative) in the presence of valinomycin. Sodium-dependent uptake kinetic constants for L-histidine were: Km=0.44 ± 0.18 mM: Vmax = 536 ± 94 nmol/mg/30 s (mean ± SD). 2-(methylamino) isobutyric acid did not inhibite L-histidine uptake. Conversely, L-histidine noncompetitively inhibited sodium-dependent uptake of 2-(methylamino)isobutyric acid and Lcysteine. L-glutamine competitively inhibited sodium-dependent L-histidine uptake. L-histidine uptake was stimulated by preloading the vesicles with either L-histidine or L-glutamine (transstimulation). L-histidine uptake was not sensitive to N-ethylmaleimide treatment but was strongly inhibited by low pH. These findings suggest that L-histidine is transported in the human placenta by a specific sodium-dependent system similar to the “N” system described in rodent hepatocytes. Furthermore, the ready availability of placental tissue may make it a useful resource for studies of human epithelial transport biology.

Histidine transfer across the human placenta: Characteristics in the isolated perfused human placenta and the effect of ethanol

Histidine is essential to normal fetal growth. In vivo, the fetal-to-maternal (F-to-M) plasma concentration ratio for histidine is the highest of any amino acid. Previously, we have shown that histidine uptake by human placental microvillous membrane vesicles (MMV) occurs by a specific, Na(+)-dependent system. In this study, we have examined the maternal-to-fetal (M-to-F) transfer characteristics of histidine, using the isolated perfused human placental cotyledon. In addition, the effect of ethanol on net transfer of histidine in this human tissue model has been assessed. During 4 h of perfusion a 1.8:1 fetal-to-maternal perfusate ratio formed for histidine. In the perfused placentae, net M-to-F transfer of histidine was saturable with an apparent Km of 0.09 mM. The perfusion experiments suggest that the F-to-M histidine gradient observed in vivo is due primarily to active transport across the placenta. The presence of 300 per cent (65 mM) ethanol in the maternal perfusate did not alter the transfer characteristics of histidine, nor that of the diffusion markers, antipyrine and L-glucose.

Calcium-Sensitive Uptake of Amino Acids by Human Placental Slices

ABSTRACT. The placenta supplies many nutrients to the fetus, including amino acids by active transport. Although the exact regulatory mechanism is unknown, a few studies have suggested a role for calcium in amino acid transport. Therefore, we examined the relationship between calcium and amino acid uptake by term human placental slices. Calcium depletion of slices significantly reduced uptake of α-aminoisobutyric (AIB), which is actively transported primarily by a sodium-dependent, carrier-mediated mechanism. Impairment of AIB uptake induced by calcium depletion was reversed by repletion with calcium but not with other divalent cations. In contrast, uptake of phenylalanine, which is transported primarily by a sodium-independent mechanism, was not affected by calcium depletion. Uptake of leucine and valine, which accumulate by both sodium-dependent and independent mechanisms, was partially affected by calcium depletion. Verapamil (10 μM), an inhibitor of transmembrane calcium flux, significantly reduced AIB uptake. Trifluoperazine, a calmodulin antagonist, also inhibited AIB uptake. Analysis of AIB uptake kinetic constants for control and calcium-depleted slices showed no change in the diffusion constant, a 37% reduction in Vmax, and a 2-fold increase in Km. The results indicate that calcium may be an important factor in the cellular regulation of active transport of amino acids.

Effect of olsalazine on sodium-dependent bile acid transport in rat ileum

Olsalazine (OLZ), a relatively new form of 5-aminosalicylic acid (5-ASA), is being used for the treatment of colitis. A major side effect of olsalazine is diarrhea, reported in 12–25% of patients. One suggested mechanism for this side effect is enhanced ileal water and electolyte secretion. We propose that OLZ may also inhibit ileal bile acid (BA) transport, resulting in choleretic diarrhea. This would result in excess BAs reaching the colon, with consequent BA-induced secretory diarrhea. Therefore, we studied the effect of OLZ on rat ileal absorption of taurocholate. BA uptake was determined in rat ileal segments, everted sacs, brush border membrane vesicles (BBMV), andXenopus laevis oocytes. Segments and everted sacs were treated with 5 mM OLZ for 30 min prior to and throughout 10-min taurocholate (Tc) uptake. Terminal ileal BBMV were used to study the effect of OLZ on sodium-dependent bile acid uptake independent of cellular metabolism. Direct effects on the bile acid carrier were examined usingXenopus laevis oocytes expressing the cloned apical rat ileal BA transporter. In ileal segments 5 mM OLZ inhibited 10-min Tc uptake by 69.4±8.8% (P<0.01) (N=10 animals). Increasing concentrations of OLZ resulted in a dose-dependent inhibition of Tc uptake. Ten-minute Tc uptake with 0.5, 1.0, 2.0, 2.5, and 5 mM OLZ was inhibited by 13.5, 39.6, 49.7, and 70.5%, respectively. In BBMV, OLZ inhibited 45-sec Tc uptake in a dose-dependent manner but did not effect Na-dependentl-alanine uptake. Kinetic analysis revealed a noncompetitive inhibition by 2 mM olsalazine. Olsalazine, 5 mM, also inhibited Na-dependent uptake of Tc into oocytes, which expressed the rat ileal sodium-dependent bile acid transporter (8.0±3.7 vs 2.6±2.0 pmol/oocyte/hr,P<0.001). OLZ inhibits sodium-dependent Tc uptake and transmucosal transport in the rat ileum in a dose-dependent manner. This inhibition is relatively specific, noncompetitive, and does not require intact cellular mechanisms. This effect of OLZ on ileal function may contribute to the diarrhea frequently observed with this drug.

Effect of N-3 polyunsaturated fatty acid supplemented diet on neutrophil-mediated ileal permeability and neutrophil function in the rat.

RATIONALE AND OBJECTIVE Fish oil, rich in n-3 polyunsaturated fatty acids, can alter leukotriene production and hence neutrophil function, factors which may be important in the inflammation of Crohns disease (CD). Therefore we studied the effect of dietary PUFA on neutrophil mediated ileal inflammation and neutrophil function in the rat. METHODS Animals were ad libitum-fed pellet diets containing 9.5% fish oil (menhaden oil, rich in n-3 PUFA) with 0.5% safflower oil, 10% safflower oil (rich in n-6 PUFA) or standard chow (6% fat) for 50 days prior to the study. Weight and circulating leukocyte and total neutrophil counts were identical in all three groups. Neutrophil mediated ileal inflammation induced by formyl-methionyl-leucyl-phenylalanine (fMLP) perfusion was evaluated by measuring macromolecular uptake of radiolabelled dextran (MW 70,000) and changes in mucosal neutrophil infiltration. RESULTS The fish oil diet group showed no difference in fMLP-induced permeability changes relative to the Chow Control group. However, the Safflower Oil supplemented diet group had a reduced permeability response (p < 0.01). Mirroring the permeability changes, there was diminished mucosal neutrophil infiltration in the Safflower Oil group following ileal perfusion with fMLP (< .005). Chemotaxis and chemiluminescence, two important neutrophil functions, were also significantly suppressed in the Safflower Oil animals (p < 0.05). CONCLUSIONS The failure of a n-3 PUFA enriched diet to diminish the ileal inflammatory response to a bacterial peptide and suppress neutrophil function in the rat suggests such therapy would not be expected to be highly successful in CD. However, it requires confirmation in man, especially under the more complicated inflammatory conditions found in CD. On the other hand, the decreased neutrophil mediated responses with a high linoleic acid (n-6 PUFA) diet warrant further investigation.

6-mercaptopurine and spermatogenesis in the young rat

In recent years, 6-MP treatment has been beneficial in the treatment of inflammatory bowel disease (IBD). Since 6-MP and its metabolites interfere with various steps in nucleic acid biosynthesis, chronic use of 6-MP could theoretically alter normal cell turnover, including spermatogenesis. Therefore, we have investigated the effect of daily 6-MP administration on spermatogenesis in the young rat. 6-Mercaptopurine was administered in clinically relevant doses 24 and 40 mg/m2. Testicular weights of rats treated with 24 mg/m2 for 75 days or 40 mg/m2 for 25 days were not significantly different among 6-MP, pair-fed, orad libitum chow-fed groups. Quantitation of the stages of seminiferous tubules or the number of homogenization-resistant, mature spermatids per testis were not affected by 6-MP treatment. In addition, 6-MP had no effect on serum testosterone or on HCG-stimulated testosterone release by the testes. These results suggest that chronic low-dose 6-MP therapy, as used in the treatment of IBD, may not carry as great a risk for suppression of spermatogenesis as theorized. Our study in animals indicates that evaluation of 6-MP and spermatogenesis in man is warranted.

Hepatic Na,K-ATPase development in the rat

The development of the enzyme Na,K-ATPase was studied in hepatic tissue from Sprague-Dawley rats at 18-20 days of gestation, 1, 3, 5, 7, 14, 21, and 30 days, and young adulthood (45 days). A developmental pattern was demonstrated for total and membrane-associated Na,K-ATPase activity. The activity in tissue homogenate, expressed per gram tissue, increased from late fetal life, 43.4 +/- 1.3 mumol Pi g liver-1 h-1, until 21 days of age, when an adult level of 187.3 +/- 19.6 mumol Pi g liver-1 h-1 was attained. A less pronounced ontogenic pattern was observed when enzyme levels were expressed as specific activity per milligram protein. The activity profile in a crude membrane preparation was similar. The potential for stimulation of enzyme activity by glucocorticoids was studied in 11-day-old animals injected with cortisone acetate (10 mg 100 g body weight-1) for 3 days. Enzyme specific activity was inducible: Specific activity was greater in cortisone-treated animals, 1.794 +/- .043 mumol Pi mg protein-1 h-1, versus controls, 1.258 +/- 0.043 mumol Pi mg protein-1 h-1 (p less than 0.01). We postulate that this developmental pattern for hepatic Na,K-ATPase activity may be a reflection of, or a contributing factor in, the ontogeny of sodium-dependent hepatic transport, such as that for bile salts.

Identification of protein kinase C-α, ϵ and ζ in rabbit ileal enterocytes

Abstract Activation of protein kinase C (PKC) has been shown to regulate electrolyte transport in rabbit small intestine. We investigated the types of PKC isoforms in rabbit ileal villus and crypt cells and the potential for phorbol 12-myristate 13-acetate (PMA) to induce translocation of the PKC from the cytosol to the plasma membrane. Our results indicate that there are at least three PKC isoforms, α, ϵ and ζ, are present in both villus and crypt enterocytes. Acute treatment with PMA resulted in translocation of the PKC-α from the cytosol to the membrane fraction in both cell types. Prolonged exposure of the villus cells to PMA resulted in a significant progressive decrement in PKC-α, suggesting down regulation. Since PMA treatment results in translocation, this isoform may be involved in the regulation of electrolyte transport in the rabbit ileum.

Amino acid uptake by the cultured human placental trophoblast: Exogenous regulation

Summary Amino acid uptake by the human placenta is known to occur via several transport mechanisms. However, regulation by extracellular factors has received relatively little attention. A recent report by this laboratory had demonstrated that α-(methylamino)isobutyric acid (MeAIB)-sensitive, α-aminoisobutyric acid (AIB) uptake by the cultured human placental trophoblast cells is stimulated by insulin. This study evaluated insulin like growth factor-1 (IGF-1) stimulation of AIB uptake in cultured human placental trophoblast cells. Na + -dependent AIB uptake was significantly stimulated by IGF-I in a time dependent manner, as early as 30 minutes. The maximum effect was at 2–4 hours of continuous exposure to IGF-I. Stimulation was dependent upon IGF-1 concentration. AIB uptake was inhibited by increasing concentrations of MeAIB. Approximately 75% of basal (unstimulated) AIB uptake was inhibited by MeAIB. The IGF-1 stimulated increment above basal AIB uptake was completely inhibited by MeAIB. At 200 ng/ml, stimulation was greater than IGF-1 than with IGF-1. The IGF-1 effect was inhibited by anti-IGF-1 receptor antibody, confirming mediation via IGF-1 receptor. In addition to the IGFs, treating the cultured trophoblast cells with 8-br-cAMP or PMA temporally stimulated AIB uptake. Therefore, Na + -dependent AIB uptake appears to be stimulated by factors which function via receptor tyrosine kinase and/or serine/threonine protein kinases (protein kinases A and C).

642 HEPATIC NA, K-ATPASE DEVELOPMENT IN THE RAT: IMPLICATIONS FOR |[ldquo]|PHYSIOLOGIC CHOLESTASIS|[rdquo]|

Na, K-ATPase (NKA) is an important enzyme for the hepatic uptake of bile acids, a process thought to be immature in neonatal liver i.e. “physiologic cholestasis”. The development of NKA activity was studied in Sprague-Dawley rats: fetal 18-20 d, newborn 1, 3, 5, 7, 10, and 14 d, and adult. NKA activity was measured in both a crude homogenate (CH) and a plasma membrane (PM) fraction, using a system containing phosphoenolpyruvate and pyruvate kinase to prevent product inhibition by ADP (Alcoholism 8:390, Am J Phys 238:E38).NKA activity in the PM fraction per gram liver was comparable to that of the CH. A similar, but less striking pattern was seen when activity was expressed per mg protein. Hepatic NKA activity shows a developmental pattern which has not reached adult levels by 14 d. The “physiologic cholestasis” of the human neonate may be due in part to insufficient Na, K-ATPase activity.