Comparison of cardiopulmonary response to endogenous nitric oxide inhibition in pigs inhabited at three levels of altitude

Nitric oxide (NO) plays an important role for the pulmonary circulation in normal and chronic hypoxia. We examined effects of endogenous nitric oxide synthase (NOS) inhibition on pulmonary and systemic vascular resistance in unanesthetized pigs living at three levels of altitude to evaluate the role of NO in adaptation to a hypoxic environment. Unanesthetized male adult pigs in three areas [Matsumoto, Japan (680 m above sea level, n=5); Xing, China (2,300 m, n=5); and Maxin, China (3,750 m, n=5)] were prepared for vascular monitoring. Pulmonary (P_pa), and systemic artery pressure (P_sa) were monitored, and pulmonary artery wedge pressure (P_cwp) and cardiac output (CO) were measured before and after treatment with a non-selective NOS inhibitor, N^w-nitro-l-argine (NLA; 20 mg/kg). Pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) were (P_pa–P_cwp)/CO and P_sa/CO, respectively. Related to altitude baseline P_pa was elevated. After NLA administration, P_pa and P_sa increased and CO decreased in all animals, resulting in increases in PVR and SVR. However, there were no significant differences in the increase in PVR and SVR in the three groups of pigs. Thus, endogenous NO production contributes to regulate the basal pulmonary vascular tone, but the development of hypoxic pulmonary hypertension appears to be independent of the NO pathway in adult pigs.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Vergleich der hämodynamischen Wirkungen vonD-Sotalol undD,L-Sotalol

Summary The hemodynamic effects of the class III drugD-sotalol and of the beta adrenergic receptor antagonistD,L-sotalol were compared in 16 rats (2 mg/kg i.v.). A group with NaCl-infusion served as control (n=10). Besides measurements in the intact circulation isovolumic registrations were performed to examine the left ventricular myocardial function without interferences.D,L-sotalol significantly reduced the dp/dt_max (61%) and the peak isovolumic left ventricular pressure (64%). It lowered the heart rate by 13% and slightly increased the mean aortic pressure to 107% (p<0.05).D-sotalol, in contrast, caused only a very transient reduction of the dp/dt_max and was comparable to the controls already 5 min after injection. The results demonstrate, that theD-enantiomer is largely avoid of the hemodynamic effects of the racemicD,L-sotalol after intravenous injection.

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Abkürzungen LVP systolischer linksventrikulärer Druck - EDP enddiastolischer linksventrikulärer Druck - AoPm mittlerer Aortendruck - peak LVSP maximaler isovolumetrischer linksventrikulärer Druck - peak dp/dtmax höchstes dp/dtmax bei isovolumetrischer Messung Mit Unterstützung der DFG     

Effects of stanozolol and L-carnitine on erythrocyte osmotic fragility during aerobic exercise in rats

The effect on erythrocyte osmotic fragility of two anabolic agents, widely used to improve physical performance, has been studied. These agents, the steroid derivative, stanozolol, and a quaternary amine,l-carnitine, were administered to rats at rest and while performing running exercises, for 12 weeks. The results show that exercise does not alter the haemolysis curve. Administration of both agents in resting condition does increase the osmotic resistance of the erythrocytes. The combined effect of drug administration and physical exercise further increases the osmotic resistance in the case of stanozolol, presumably due to the steroid-erythrocyte membrane interaction as well as by an erthyropoietic effect.     

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.     

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.     

N-Acetyl-l-cysteine and its derivatives activate a Cl− conductance in epithelial cells

N-Acetyl-l-cysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders including cystic fibrosis (CF). The beneficial effects of NAC are empirical and the exact mechanism of action in the airways remains obscure. In the present study we examined the effects on whole-cell (we) conductance (G _m) and voltage (V _m) of NAC and the congeners S-carboxymethyl-l-cysteine (CMC) andS-carbamyl-L-cysteine (CAC) andL-cysteine in normal and CF airway epithelial cells.L-Cysteine (1 mmol/1) had no detectable effect. The increase inG _m (G_m) by the other compounds was concentration dependent and was (all substances at 1 mmol/1) 3.8 ± 1.4 nS (NAC; n = 11), 4.2 ± 1.0 nS (CMC;n = 16) and 3.8 ± 1.6 nS (CAC;n = 18), respectively. The changes in G_m were paralleled by an increased depolarization (V_m) when extracellular Cl^– concentration was reduced to 34 mmol/1: under control conditions = -4.1 ± 2.1 versus 10.2 ± 2.1 mV in the presence of NAC, CMC, CAC (n = 36). In the presence of NAC, CMC and CAC, the reduction in Cl^– concentration was paralleled by a reduction ofG _m by 2.1 ± 0.4 nS (n = 35), indicating that all substances acted by increasing the Cl^– conductance. Analysis of intracellular pH did not reveal any changes by any of the compounds (1 mmol/1). A Cl^– conductance was also activated in HT₂₉ colonic carcinoma and CF tracheal epithelial (CFDE) cells but not in CFPA1 cells, which do not express detectable levels of F508-CFTR, suggesting that the presence of CFTR may be a prerequisite for the induction of Cl^– currents. Next we examined the ion currents in Xenopus oocytes microinjected with CFTR-cRNA. Water-injected oocytes did not respond to activation by forskolin and 3-isobutyl-l-methylxanthine (IBMX) (Gm = 0.08 ±0.04 S;n = 10) and no current was activated when these oocytes were exposed to NAC or CMC. In contrast, in CFTR-cRNA-injected cocytesG _m was enhanced when intracellular adenosine 3,5-cyclic monophosphate (cAMP) was increased by forskolin and IBMX (G _m = 4.5 ± 1.3 S;n = 8).G _m was significantly increased by 0.74 ± 0.2 S (n = 11) and 0.46 ± 0.1 S (n = 10) when oocytes were exposed to NAC and CMC, respectively (both I mmol/1). In conclusion, NAC and its congeners activate Cl^– conductances in normal and CF airway epithelial cells and hence induce electrolyte secretion which may be beneficial in CF patients. CFTR appears to be required for this response in an as yet unknown fashion.     

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     

Residual Stress Patterns Affect Cell Distributions on Injection-Molded Poly-<Emphasis Type="SmallCaps">l</Emphasis>-Lactide Substrate

The effects of residual intra-substrate stress distribution on cell behavior have not been systematically investigated. Thus, the objective of this research was to analyze the relationship between cell distribution and internal stress patterns. A photoelastic method was used for residual stresses identification. Poly-l-lactide (PLLA) discs were prepared using an injection molding technique. MG-63 and NIH-3T3 cells were cultured on the surface of the PLLA disc. The cell distributions for high and low-stress regions were measured and compared. There were significantly more cells in the low-stress regions relative to high-stress analogs (p < 0.05). Further, linear relationships were demonstrated for both MG-63 and NIH-3T3 models with high correlation coefficients of 0.80 and 0.95, respectively. These results suggest that the distribution of residual stress in substrates affect cell behavior. These findings may provide greater insight into the interaction between cells and substrates, and may serve as a useful reference in future clinical study.     

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]     

Evaluation of chondrocyte growth in the highly porous scaffolds made by fused deposition manufacturing (FDM) filled with type II collagen

Highly porous poly(d,l-lactide-co-glycolide) (PLGA) scaffolds for cartilage tissue engineering were fabricated in this study using the fused deposition manufacturing (FDM) process and were further modified by type II collagen. The average molecular weight of PLGA decreased to about 60% of the original value after the melt-extrusion process. Type II collagen exhibited sponge-like structure and filled the macroporous FDM scaffolds. An increase of the fiber spacing resulted in an increase of the porosity. The storage modulus of FDM scaffolds with a large fiber spacing was comparable to that of the native porcine articular cartilage. Although the FDM hybrid scaffolds were swollen in various extents after 28 days of in vitro culture, the seeded chondrocytes were well distributed in the interior of the scaffolds with a large fiber spacing and neocartilage was formed around the scaffolds. The study also suggested that a low processing temperature may be required to produce PLGA precision scaffolds using FDM.