Renal handling ofl-histidine studied by continuous microperfusion and free flow micropuncture in the rat

Renal tubular reabsorption ofl-histidine (His) was measured in vivo et situ by continuous microperfusion and free flow micropuncture of single proximal convoluted tubules of the rat kidney. The reabsorption is shown to be saturable. A permeability coefficient (P) of <29 m² · s^–1, a maximum reabsorption rate (J _max) of 2.75±1.05>J _max>1.97±0.86 (SEM) nmol · m^–1 · s^–1 and an affinity constant (K _m) of 13.8±4.2>K _m>10.9±4.0 (SEM) mol · l^–1 (lower values forP=29 m² · s^–1, higher values forP=0) were calculated from the microperfusion data. Using these constants and taking backflux of His and water reabsorption into account a good fit with the concentration profile of His along the proximal tubule — measured by free flow micropuncture — was obtained.Varying the buffered pH-values of the perfusion fluids (5.0 or 7.4) influenced neither the active reabsorption nor passive permeability of His. This indicates that the charge of the imidazol group of His does not play a significant role in His reabsorption. Further experiments showed that the addition of 20 mmol · l^–1 l-arginine — a strong inhibitor of the reabsorption system for dibasic ammino acids — did not have a significant effect on the reabsorption ofl-histidine. It is concluded, therefore, that His is reabsorbed by a system for neutral amino acids. Non ionic diffusion does not play an important role for His reabsorption.Part of this work was presented at the 51st meeting of the German Physiological Society in Kiel, 1979 [15]     

Micropuncture and microperfusion study ofl-glucose secretion in rat kidney

Summary Studies with the free flow micropuncture technique have shown that the ratio of TF/P_l-glucose to TF/P_Inulin in proximal tubular fluid, in distal tubular fluid, and in more than half of the final urine samples measured was greater than one, which suggests thatl-glucose was actively secreted. Studies with the microperfusion technique confirmed this finding and showed thatl-glucose was secreted by the proximal tubules. A maximum rate of secretion was reached at a plasma concentration of 4 mM. The tubular secretion ofl-glucose was augmented by the presence of 16.6 mMd-glucose in tubular lumen and inhibited by 10^–4 M phlorizin. Kinetic analysis showed that theV _max values forl-glucose secretion in the absence and in the presence ofd-glucose are 5.0×10^–10 and 6.3×10^–10 mol×cm^–2×sec^–1 respectively which were very close to the value reported for theV _max ford-glucose reabsorption. However, theK _m forl-glucose secretion was 3.1 mM and was reduced to 1.6 mM whend-glucose was present in the perfusion fluid. TheK _m ford-glucose reabsorption has been reported to be 0.6 mM (8). The results of this investigation were interpreted as being consistant with the hypothesis thatl-glucose secretion andd-glucose reabsorption share the same carrier system.On sabbatical leave from Univ. Louisville, School of Medicine and supported by a NIAMD Special Fellowship (1-F3-AM-32,720-01) and a research grant of USPHS (AMO2217-10).     

Contraluminal transport of hexoses in the proximal convolution of the rat kidney in situ

In order to study contraluminal hexose transport, concentration and time-dependent influx of³H-2-deoxy-d-glucose from the interstitium into cortical tubular cells has been measured. The influx curves fit to a two parameter kinetics (K _m 1.3±0.2 mmol/l,J _max 0.67±0.16 pmol/s · cm) plus an additional diffusion term (withP=6·10^–8 cm²/s) and a distribution ratio extracellular to intracellular amount of 2-deoxy-d-glucose of 10.6. Since the extracellular to intracellular free water space as estimated from morphological data was 12, one must conclude that glucose has only free access to 1/3 of the cell water. The intracellularly accessible space was augmented when the tubules were preperfused for 10 s with hypotonic saline. Thereby an increase of the compartment into which diffusion occurs was revealed and a final rupture of this intracellular compartment at 1/4 isotonic solutions was observed. Total replacement of ions in the peritubular perfusate by mannitol did not change 2-deoxy-d-glucose influx, indicating that it is Na⁺-independent. By adding isotonic concentrations of the respective sugars to the capillary perfusate, three degrees of inhibition of 2-deoxy-d-glucose influx could be revealed: strong inhibition byd-glucose, methyl--d-glucoside,d-mannose, 3-O-methyl-d-glucose, 2-deoxy-d-galactose, methyl--d-galactoside and 6-deoxy-d-glucose, moderate inhibition byd-galactose,l-glucose,l-mannose andd-fructose, no or borderline inhibition by methyl -d-glucoside, 2-deoxy-methyl--d-galactoside, 1-thio--d-glucose, 1-thio--d-galactose, 5-thio--d-glucose, myo-inositol and mannitol. The contraluminal 2-deoxy-d-glucose influx was also inhibited by phloretin, chlormerodrin and preperfusion with cytochalasin B. Starvation as well as streptozotocin diabetes has no influence on contraluminal 2-deoxy-d-glucose transport. Thus, in contrast to the luminal hexose transport system the contraluminal system is Na⁺-independent, does not require on OH-group at C-atom 2, acceptsl-glucose and fructose, but not an -methyl group at C-atom 1.     

Polar distribution of sodium-dependent and sodium-independent transport system forl-lactate in the plasma membrane of rat enterocytes

The uptake ofl-lactate by rat small intestinal brush-border and basal-lateral plasma membrane vesicles has been studied.l-Lactate uptake by the isolated membrane vesicles is osmotically sensitive and represents predominantly transport into an intravesicular space and not binding to the membranes.The transport ofl-lactate across the brush-border membrane is stimulated by sodium, whereas the transport across the basal-lateral plasma membrane is sodium-independent. In both types of membrane vesiclesl-lactate is transported faster thand-lactate andl-lactate transport is inhibited by -cyano-cinnamic acid.l-Lactate transport across basal-lateral membranes is inhibited byd-lactate and pyruvate and transstimulated byl-lactate and pyruvate.The polar distribution of transport system forl-lactate in the plasma membrane of rat enterocytes—a Na⁺/l-lactate cotransport system in the brush-border membrane and a facilitated diffusion system in the basal-lateral membrane — can explain the fact that in the intact epitheliuml-lactate produced by cell metabolism is preferentially released on the serosal side and could enable the cell to perform vectorial, secondary active transport ofl-lactate from the intestinal lumen to the serosal compartment.     

Fictive locomotion in the adult decerebrate rat

In adult immobilised, decerebrate rats, administration of l-3,4-dihydroxyphenylalanine, stimulation of the mesencephalic locomotor centre, or a combination of the two elicited fictive locomotor patterns in hindlimb muscle nerves. The patterns correspond closely to those observed in decerebrate animals that were free to move.     

Rat proximal tubule D-glucose transport as a function of concentration,flow, and radius

Summary From earlier microperfusion studies ofD-glucose and water reabsorption in the proximal surface nephron of the rat,D-glucose was found to be removed by a saturable carrier and by an apparent coupling with net fluid reabsorption. Equations appropriate to describe this system were developed. They incorporated carrier-mediatedD-glucose transport, net water transport, and water-coupled solute transport. Water reabsorption was assumed to be constant either per unit surface area, or per unit volume of the nephron, and the rate of carrier-transportedD-glucose was assumed constant per unit length, per unit surface area, or per unit volume of the tubule. The possibility thatD-glucose could be reabsorbed via two carrier systems was also explored analytically. It was observed from this treatment that the fraction ofD-glucose reabsorbed would change if the perfusion rate was changed. With an increase in perfusion rate, a decrease in reabsorbed fraction was seen which indicates that if net fluid reabsorption is proportional to volume, carrier-mediated sugar transport is proportional to surface area or length of the tubule. From these relationshipsJ _max, the maximal rate of carrier-transported sugar, was calculated to be 3.3×10⁻¹⁰M/cm² sec, a value comparable to that reported from other laboratories. The results of this analysis are compatible with the data obtained both by micropuncture experiments during free flow and by glucose clearance studies until theT _mG is reached. The possibility that theT _mG obtained in clearance studies is due to a decrease in the fraction of fluid reabsorbed in the proximal tubule or to a second saturable carrier is discussed. It is observed that, in either case, if load is increased by increasing the glomerular filtration rate, noT _mG would be reached, or stated another way, one would predict from the analysis thatT _mG would be proportional to glomerular filtration rate. Partially supported by NIH-AM-10779-02 and “Deutsche Forschungsgemeinschaft”. Partially presented at the XXIV International Congress of Physiological Societies, Washington, D.C., 1968. PHS post doctoral fellow 1-F2-AM-37056-01 and Dr. Henry C. and Bertha H. Buswell Fellow Dr. Henry C. and Bertha H. Buswell Fellow     

The stimulus-secretion coupling of amino acid-induced insulin release

l-Glutamine enhances insulin release evoked byl-leucine in isolated rat pancreatic islets. The enhancing action ofl-glutamine, which is a rapid but steadily increasing and not rapidly reversible phenomenon, is not attributable to any major change in either K⁺ or Ca²⁺ outflow from the islet cells. It coincides with an apparent increase in Ca²⁺ inflow rate and, hence, with Ca accumulation in the islets. The initial ionic response tol-leucine is not qualitatively altered by the presence ofl-glutamine. In their combined capacity to stimulate⁴⁵Ca net uptake in the islets,l-glutamine can be replaced byl-asparagine but not byl-glutamate, whereasl-leucine can be replaced byl-norvaline orl-isoleucine, but not byl-valine, glycine orl-lysine. Such a specificity is identical to that characterizing the effect of these various amino acids upon insulin release. It is postulated that the release of insulin evoked by the combination ofl-leucine andl-glutamine involves essentially the same remodelling of ionic fluxes as that evoked by other nutrient secretagogues with, however, an unusual time course for the functional response tol-glutamine.     

The effects of exogenous amino acids on the relaxant responses of pig urethral smooth muscle evoked by stimulation of the inhibitory nitrergic nerves

Inhibitory innervation of urethral smooth muscle is mediated partly through release of NO. We investigated the mechanisms involved in the supply of the substrate l-arginine to NO synthase by examining the relaxant response of the muscle to electrical field stimulation (EFS) and the effects of addition of amino acids to the bathing medium. Relaxant responses persisted during hours of repetitive stimulation but were enhanced rapidly by addition of l-arginine (the arginine paradox). Addition of l-lysine (competes with l-arginine for transport on the y⁺ carrier) and l-glutamine (competing on the y⁺L carrier) attenuated the enhancement. Enhancement persisted after washing but was reversed by application of l-lysine, suggesting that exogenous l-arginine fills an intracellular pool and that l-lysine can trans-stimulate its efflux from the pool. After prolonged depolarization in high-K⁺, Na⁺-free solution the relaxant response became purely nitrergic. Addition of l-arginine during the exposure continued to enhance the subsequent responses but l-glutamine added with l-arginine, could no longer reduce this enhancement. The results show the arginine paradox in inhibitory nerves and suggest the involvement of y⁺ and y⁺L carriers in the transport of l-arginine.     

Molecular specificity of tubular reabsorption ofl-proline

In microperfusion experiments the reabsorption of³H and¹⁴C labelledl-proline by two recently defined transport systems (one with high capacity and low affinity, the other one having the opposite characteristics) was measured in vivo et situ on addition of several amino acids and some N-methylated derivatives.The high capacity system is apparently an unspecific system for neutral amino acids. The methylation of the amino group does not change the affinity to the system. The affinity decreases in the order phenylalanine >glutamine>alanine>proline, hydroxyproline >glycine.The low capacity system seems to be a specific reabsorption mechanism for imino acids like proline, hydroxyproline, sarcosine and N-methylalanine. Common neutral amino acids are not accepted.The different characteristics of both transport systems are also demonstrated by the finding that the affinity of phenylalanine for the high capacity system is about 5 times higher but its affinity for the low capacity system is about 50 times lower than the affinity for proline.Parts of this work were presented at the 50th Meeting of the German Physiological Society at Göttingen, FRG, October 1978 [26]     

The Na+-dependent binding of [3H]l-aspartate in thaw-mounted sections of rat forebrain

Binding of [³H]l-aspartate to thaw-mounted coronal sections of frozen rat forebrain was strong in grey regions of telencephalon (neocortex, hippocampus and neostriatum), but it was weaker and unevenly distributed in diencephalon. At low nanomolar concentrations of ligand used in the present studies, [³H]l-aspartate binding was strongly inhibited by l-threo-3-hydroxyaspartate and l-trans-pyrrolidine-2,4-dicarboxylate, compounds known to be substrate/inhibitors of the high affinity uptake of l-glutamate and l-aspartate. None of the typical ligands for the glutamate and aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), N-methyl-d-aspartate and kainate, produced a strong enough inhibition (only CNQX at 100 M weakly inhibited) of the Na⁺-dependent [³H]l-aspartate binding to suggest that [³H]l-aspartate was bound to the receptor binding sites. Furthermore, the binding was absolutely dependent on the presence of Na⁺ in the incubation medium. It is concluded that [³H]l-aspartate is a ligand suitable for autoradiographic studies of the distribution of Na⁺-dependent, high affinity uptake of acidic amino acids in the central nervous system (CNS). However, feasibility of using [³H]l-aspartate as a specific marker of glutamatergic and/or aspartergic synapses in the CNS requires further investigation.     

Mutation and haplotype analyses of the <Emphasis Type="Italic">MUT</Emphasis> gene in Japanese patients with methylmalonic acidemia

Methylmalonic acidemia (MMA) is caused by a deficiency in the activity of l-methylmalonyl-CoA mutase (MCM), a vitamin B12 (or cobalamin, Cbl)-dependent enzyme. Apoenzyme-deficient MMA (mut MMA) results from mutations in the nuclear gene MUT. Most of the MUT mutations are thought to be private or restricted to only a few pedigrees. Our group elucidated the spectrum of mutations of Japanese mut MMA patients by performing mutation and haplotype analyses in 29 patients with mut MMA. A sequence analysis identified mutations in 95% (55/58) of the disease alleles. Five mutations were relatively frequent (p.E117X, c.385 + 5G > A, p.R369H, p.L494X, and p.R727X) and four were novel (p.M1V, c.753_753 + 5delGGTATA, c.1560G > C, and c.2098_2099delAT). Haplotype analysis suggested that all of the frequent mutations, with the exception of p.R369H, were spread by the founder effect. Among 46 Japanese patients investigated in the present and previous studies, 76% (70/92) of the mutations were located in exons 2, 6, 8, and 13. This finding – that a limited number of mutations account for most of the mutations in Japanese mut MMA patients – is in contrast with results of a previous study in Caucasian patients.     

Fabrication and evaluation of microgrooved polymers as peripheral nerve conduits

Cell alignment plays an important role in the repair of damaged peripheral nerves. The aligned Schwann cells could direct the axonal outgrowth during nerve reconstruction. One way of aligning Schwann cells is to use surface grooves in micrometric dimensions. In this study, microgrooves on chitosan or poly(d,l-lactide) (PLA) were fabricated and the behaviors of Schwann cells and glial cell line C6 on these surfaces were examined. It was found that Schwann cells and C6 cells could be successfully aligned by the microgrooves, and express the genes related to the production of neurotrophic factors. The polymer conduits with microgrooves on the inner surface were implanted in rats to repair the damaged sciatic nerve. The microgrooved conduits were demonstrated to enhance peripheral nerve regeneration as compared to the smooth conduits.     

Renal tubular reabsorption of 1,5-anhydro-D-glucitol and D-mannose in vivo in the rat

1,5-Anhydro-D-glucitol (AG) is efficiently reabsorbed in renal tubuli by a mechanism that is saturated at high AG concentrations. To gain insight into the stereospecific requirements of the mechanism, we employed an in vivo loading test technique in which rats were injected with anhydrosugars and aldohexoses in doses that led to excretion of the sugar injected, thus saturating tubular reabsorption. Administration of AG elicited an increase in the excretion of D-mannose (P<0.0005), while D-mannose caused AG to appear in urine. Administration of 1,5-anhydro-D-mannitol led to increased excretion of D-mannose (P<0.0005) and the appearance of AG in urine. The effects of 1,5-anhydro-D-mannitol on the excretion of D-mannose and AG, and the effect of D-mannose on AG were dependent on the dose. Myoinositiol, mannitol and C-3–C-6 epimers of AG did not interfere with the reabsorption. The mechanism was highly phlorizin-sensitive. Repeated administration of 1,5-anhydro-D-mannitol rapidly depleted the rat organism from mobilizable AG. The AG space calculated (53% of body weight) suggested the presence of considerable cellular stores of AG. D-Mannose and AG are regular components of the plasma monosaccharide profile. The data suggest that the two sugars are reabsorbed in renal tubuli by a common mechanism, which is distinct from the main D-glucose reabsorption system. The presence of a glucose-type C-3–C-6 and pyranose structure is required for a sugar to be transported by the system. The mechanism accommodates both an axial and an equatorial hydroxyl group at C-2, but a hydroxyl group at C-1 is not required.     

Virtual elimination of the interference of unstirred water layers on intestinal sugar transport kinetics by use of the tissue accumulation method at appropriate shaking rates

The intestinal transport of sugars and amino acids seems to follow Michaelis-Menten kinetics, but the presence of unstirred water layers at the outer face of the brush border membrane may distort kinetic measurements. According to current theory, the capacity parameter,J _mc ^max would not be affected, but theK _t would be increased to a higher value,K _t , in proportion to the thickness of the unstirred water layer,d.We reasoned that by increasing the shaking rate in the tissue accumulation method,d might drop to such small values thatK _t would fall to a constant level practically equal to the trueK _t .We measuredd-galactose influx into rings of everted hamster intestine as a function of both the substrate concentration and the shaking rate. Our results show that as the circular stirring rate increases from 0.38–6.2 Hz,J _mc ^max remains constant, as expected, butK _t first drops, then levels off to reach a plateau between 2 and 6.2 Hz. We conclude that the averageK _t values in this frequency range (K _t =7.4 mM) represent the true transportK _t . Furthermore, all previous kinetic work performed in our laboratory has been carried out under identical conditions, including shaking rates of 4 Hz. The validity of our preceding results is thus upheld.     

Amino acid-evoked membrane potential and resistance changes in pancreatic acinar cells

The effect of some neutrall-amino acids, alanine, valine and proline, on the pancreatic acinar cell membrane potential and resistance was investigated. Simultaneous recordings were made with two intracellular microelectrodes on isolated superfused segments of mouse pancreas. Amino acids were applied by inclusion at known concentrations in the superfusion fluid or by microionophoresis from extracellular micropipettes. l-Alanine (10 mmol·l^–1) evoked a maximal membrane depolarization of about 18 mV. A just detectable depolarization was observed at 0.1 mmol·l^–1 (3 mV). Halfmaximal depolarization was observed at 1.6 mmol·l^–1.d-Alanine had virtually no effect.Microionophoretic applications ofl-alanine,l-valine orl-proline evoked depolarization and resistance reduction with a very short delay (<50 ms). The dose response curves for depolarization and resistance reduction were similar.The amplitude of the depolarization evoked byl-alanine,l-valine andl-proline depended linearly on the level of the pre-set membrane potential (membrane potential could be changed by direct current injection). With decreasing intracellular negativity there was a decrease in the size of the amino acid-evoked depolarization. When the membrane potential was inside positive the amplitude became very small. Extrapolation of the linear relations between membrane potential and size of depolarization revealed a null potential of +20 to +45 mV.Thel-alanine-evoked depolarization was acutely reduced but not abolished by replacing extracellular Na by Tris or Li. l-Alanine,l-proline andl-valine exhibited mutual inhibition of evoked depolarization even when the depolarizing effect of the first applied amino acid was balanced by direct current injection.It is concluded that severall-amino acids act on the pancreatic acinar plasma membrane by opening conductance pathways mainly permeable to Na.     

Role of poly-l-lysine-coated plates and fetal calf serum concentration in sheep chondroprogenitor cell culturing

Conventional methods for differentiation of chondroprogenitor cells on plastic plates face several problems that hinder the application of this method for the treatment of chondrogenic injury. This work focused on the effect of poly-l-lysine (PLL)-coated plastic surfaces and fetal calf serum concentration on the chondroprogenitor cells. In the present study, cartilage was isolated from the articular cartilages of sheep and the cells were seeded on PLL-coated plates in various serum concentrations. Histochemical analysis was used to determine chondrogenic differentiation of the cells. According to our results, the cells formed three-dimensional masses and chondrogenic cells. In the present investigation, the best culture conditions for maximum proliferation of isolated cells were examined. Taken together, the results indicated that PLL may have some effect on the adhesive properties of chondroprogenitor cells and could be used for cartilage engineering. The first two authors contributed equally to this work.     

Insulin enhances l-dopa renal proximal tubule uptake: a regulatory mechanism impaired in insulin resistance

A stimulatory role for insulin in the uptake of neutral amino acids has been reported for a variety of tissues. Here we examine the effect of insulin on l-dopa uptake by proximal tubule cells (PT cells) isolated from control and fructose-fed rats (FR-rats, 10% w/v fructose solution in tap water), a model of insulin resistance. Insulin (200 U/ml) increased l-dopa uptake into PT cells by about 50% (705±186 vs.1117±140 pmol l-dopa/mg protein per minute) (p<0.05). The higher uptake correlated with a 40% increase in the number of high-affinity l-dopa transport sites (l-dopa 0.2 M) (0.59±0.05 vs. 0.82±0.09 pmol l-dopa/mg protein per minute), without changing their affinity. The effect of insulin was not modified by ouabain (1 mM), nocodazole (1–10 M) or colchicine (50–100 M), whereas it was abolished by cytochalasin D or latrunculin B (both 1 M). This suggests that the process is independent of Na⁺,K⁺-ATPase activity or the microtubule network but that it requires the integrity of the actin cytoskeleton. l-dopa transport by the low-affinity transport sites (l-dopa 5 M) was not affected by insulin, neither was the effect of insulin observed in PT cells isolated from FR-rats. In line with this, FR-rats showed lower renal l-dopa reabsorption as compared to control animals (81±4 vs. 51±9%). Taken together, our results support the involvement of insulin in the multifactorial regulation of renal l-dopa reabsorption.     

Facilitated diffusion of lactic acid in the guinea-pig placenta

In the guinea-pig placenta which was artificially perfused on the fetal side while maternal placental blood flow was controlled, the placental transfer per mean transplacental concentration difference (the transfer coefficient TC) was determined for lactate. TC forl-lactate (TC_LL) was compared to that ford-lactate (TC_DL) and measured for various concentrations ofl-lactate, bicarbonate, pyruvate and CO₂. Applying a closed circuit perfusion technique,l-lactate and proton concentrations on both sides of the placenta were followed during infusion of HCl and sodiuml-lactate into the fetal circulation.It was found that TC_LL is 3 times TC_DL. TC_LL is depressed by increasing concentrations ofl-lactate while TC for Cl-36 is not. TC_LL is also depressed by 50 mmol·l^–1 pyruvate. Concentration changes of glucose do not affect TC_LL. TC_LL rises with the proton concentration, independently of the concomitant changes of the bicarbonate concentration. Transplacentral proton concentration gradients producel-lactate concentration gradients and vice versa.It is concluded that (1) facilitated diffusion ofl-lactate occurs in the placenta and that (2)l-lactate transfer is coupled with proton transfer. Beside the well-known placental transport system for glucose this is the second passive transport system found in a placenta.Supported by the Deutsche Forschungsgemeinschaft (Mo 105/8)Partly presented at the Frühjahrstagung der Deutschen Physiologischen Gesellschaft, 1977     

Gene roles in the prn cluster of Aspergillus nidulans

Summary The roles of the four genes of the prn gene cluster involved in L-proline catabolism in Aspergillus nidulans have been investigated. prnD and prnC encode, respectively, proline exidase and ^I-pyrroline-5-carboxylate (P5C) dehydrogenase. prnB is almost certainly the structural gene for the proline-inducible major proline permease. The prnA product has no structural role in these activities but is a positive acting regulatory molecule necessary for the expression of prnD, prnC and, to a lesser extent, prnB. Evidence favouring de novo synthesis of P5C dehydrogenase upon induction in the presence of a functional prnA allele is also presented.