Potassium transport in epithelial cells isolated from small intestine of the chicken

The transport of potassium has been studied in epithelial cells isolated from chicken small intestine using⁸⁶Rb as a tracer for K⁺. (i) The uptake studies revealed that about 60% of the total K⁺ net flux is inhibited by ouabain and therefore mediated by the Na⁺–K⁺-ATPase. About 20% of the ouabain-insensitive K⁺ net influx was inhibited by furosemide, bumetanide and by either Na⁺ or Cl^– removal from the incubation solution, suggesting that a Na⁺/Cl^–/K⁺ cotransport system might be present in chicken enterocytes. (ii) The efflux of K⁺ was measured from cells preloaded with⁸⁶Rb. K⁺ efflux was inhibited by Ba²⁺, quinine and verapamil; it was stimulated by A23187, and it was unaffected by 3,4-diaminopyridine. Apamin, that has no effect on basal rates of K⁺ efflux, abolished the effect of A23187. These findings suggest that K⁺ efflux appears to occur through Ca²⁺-activated K⁺ channels.     

Excitatory amino acid response in isolated spiral ganglion cells of guinea pig cochlea.

1. The physiological and pharmacological properties of excitatory amino acid (EAA)-induced responses were investigated in acutely isolated spiral ganglion cells of guinea pig by a conventional patch-clamp technique combined with a rapid drug application (Y-tube) method. 2. L-glutamate (Glu) and its agonists, quisqualate (QA) and kainate (KA), induced inward currents in a concentration-dependent manner at a holding potential (VH) of -70 mV. The values of half-maximal concentration (EC50) were 4.0 x 10(-4) M for Glu, 2.3 x 10(-5) M for QA, and 1.4 x 10(-4) for KA. The Hill coefficients were 0.96, 1.00, and 1.56 for Glu, QA, and KA, respectively. However, one of Glu agonists, N-methyl-D-aspartate (NMDA), and another excitatory amino acid, L-aspartate (Asp), did not induce any responses even in Mg2(+)-free external solution containing 10(-6) M glycine (Gly). 3. The current-voltage (I-V) relationships for the Glu-, QA-, and KA-induced responses were linear, and these reversal potentials were near 5 mV. 4. Kynurenic acid (Kyn), 6,7-dichloro-3-hydroxy-2-quinoxalinecarboxylic acid (diCl-HQC), and quinoxalinediones such as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitro-quinoxaline-2,3-dione (DNQX) suppressed the Glu-, QA-, and KA-induced currents in a concentration-dependent manner. The inhibitory potency was in the order of DNQX = CNQX greater than diCl-HQC greater than Kyn. 5. CNQX antagonized the Glu-, QA-, and KA-induced currents without affecting the maximum responses showing no voltage-dependency, indicating the competitive inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of amino acid transport in hepatocytes isolated from adult and old rats

The 2-aminoisobutyric acid transport in hepatocytes isolated from 3- and 24-month-old rats was studied and some age-related differences were observed. The basal uptake appeared to be almost constant in cells from old animals during the incubation time, while, in the cells from adults, it showed a progressive increase, interpreted as being due to a derepression mechanism. Epinephrine and glucagon increased the transport in hepatocytes from animals of both ages, even if with a slightly different pattern; the hormones increased the Vmax, while the Km was unchanged at each age tested. However, the glucagon-induced increase in Vmax was lower in the older animals. The mechanism of hormonal action appeared to be similar in adult and old rats. In fact the uptake stimulation by glucagon and epinephrine showed a dependence on protein synthesis. The epinephrine effect was mediated by alpha-adrenergic receptors. No effect was exerted by extracellular amino acids on hepatocytes from 24-month-old animals, suggesting a loss of adaptative regulation mechanism with aging. This behaviour was reflected in the kinetic parameters; in fact the Vmax was not modified by extracellular amino acids at 24 months of age, while it appeared to be strongly decreased in the adult.     

Effect of polyinosinic-polycytidylic acid on the number of colony-forming cells in the hematopoietic organs of guinea pigs

An increase in the number of cells forming discrete colonies in monolayer cultures of myelokaryocytes (colony-forming cells) was observed in guinea pigs 8–24 h after receiving an intraperitoneal injection of polyinosinic-polycytidylic acid in a dose of 300μg/kg. The number of colony-forming cells in the spleen was reduced at these same time after injection of the compound. After 3 days the number of colony-forming cells in the spleen increased, but in the bone marrow it decreased. These values returned to normal 14 days after injection of the compound. The changes observed are evidently explainable on the grounds that the compound has a marked effect on the fraction of colony-forming cells in hematopoietic organs that are stromal elements of the stem-cell type.     

Effect of triiodothyronine on system A amino acid transport in cells of rat submandibular gland

The effect ofL-3,5,3-triiodothyronine (T₃) on -aminoisobutyric acid (AIB) transport in isolated cell suspensions of rat submandibular gland was investigated. The uptake of ATB by these cells appeared to require extracellular Na⁺ and was inhibited by ouabain (10^–3 M). Cell suspensions from thyroidectomized rats which have been given three successive doses of T₃ on alternate days (50 g/100 g BW) showed a significant increase in AIB uptake compared with cells isolated from thyroidectomized rats treated with diluent. Efflux of AIB from the cell suspension was not affected by T₃. There was no significant changes in AIB uptake 12 h after a single injection of T₃ (50 g/100 g BW). However, there was a significant 49% and 65% increase in AIB net uptake at 24 and 48 h, respectively, after T₃ treatment. Under similar conditions, the cell suspension showed a 48% increase in NaK-ATPase activity at 12 h and to a peak of 61% at 24 h. Therefore, changes in NaK-ATPase activity preceded the changes in AIB net uptake upon treatment with T₃, implying that AIB uptake is probably mediated, at least in part, by the activity of NaK-ATPase.Abbreviations AIB -Aminoisobutyric acid - HBBS Hank's buffer salt solution - T₃ triiodothyronine This study was supported by NIH grant (AM 28590) and USUHS grant CO 7623     

Excitatory amino acid receptors on isolated retinal ganglion cells from the goldfish.

1. Whole-cell currents activated by the excitatory amino acids L-glutamic acid (glutamate, Glu), L-aspartic acid (Asp), and their analogues N-methyl-D-aspartate (NMDA), kainic acid (KA), quisqualic acid (QA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were recorded from ganglion cells enzymatically dissociated from goldfish retina and grown in culture. All agonists induced detectable whole-cell responses in the majority of cells cultured from 2 to 72 h. 2. Currents activated by each of the agonists were selective for cations (Na+) over anions (Cl-). The responses to Glu, NMDA, and Asp were each potentiated when 1 microM glycine was coapplied. Extracellular Mg2+ blocked completely the response to NMDA plus glycine in cells held at negative potentials, but the block was relieved when cells were more depolarized. 3. Dose-response measurements revealed a rank order of sensitivity to the Glu analogues in the presence of 1 microM glycine and zero extracellular Mg2+; QA greater than AMPA greater than NMDA greater than KA. Cells were not responsive to APB (L-2-aminophosphonobutyric acid). 4. Kynurenic acid (Ky) produced a noncompetitive block of the NMDA response in the presence of 1 microM glycine with a Ki of 40 microM. Responses to KA, QA, and AMPA were blocked competitively by Ky with Kis of 72, 148, and 656 microM, respectively. QA and AMPA competitively blocked the response to KA with Kis of 114 microM and 1 mM, respectively. NMDA single channels had a mainstate slope conductance of 27-30 pS and two subconductance levels of 5 and 24 pS at 13 degrees C in symmetrical Na+. 5. Whole-cell responses to QA and AMPA were highly correlated, suggesting that QA and AMPA activated the same receptor or class of receptors; whereas, responses to QA and KA were not well correlated, suggesting that these agonists at least in part activated separate receptor populations.     

Metabolism of platelet-activating factor in neutrophils isolated from Pichinde virus-infected guinea pigs.

Metabolism of platelet-activating factor (PAF) was studied in cultured polymorphonuclear neutrophils (PMNs) obtained from Pichinde virus-infected strain 13 guinea pigs. Neutrophils obtained from control and infected guinea pigs on postinoculation days 10 and 14 were incubated with [3H]lyso-PAF, [3H]PAF, or [3H]acetate. After incubation for 1 h at 37 degrees C, formation of [3H]acyl-PAF from either [3H]lyso-PAF or [3H]PAF increased significantly in PMNs from infected guinea pigs compared to control PMNs. Furthermore, total radioactivity from [3H]lyso-PAF or [3H]PAF was higher in PMNs from infected animals than in those from controls. However, compared to PAF production by control PMNs, production of PAF by infected PMNs was unchanged. These results suggest that PMNs may not be the major source of increased blood PAF levels during Pichinde viral disease.     

Alpha-aminoadipic acid blocks the Na+-dependent glutamate transport into acutely isolated Müller glial cells from guinea pig retina

The effect of the glial toxin α-aminoadipic acid (AAA) upon theNa⁺/glutamate cotransporter of acutely isolated guinea pig retinal glial cells was studied using the whole-cell voltage-clamp technique. Glutamate evoked an in ward current in these cells at negative holding potentials dependent on the presence of extracellular Na⁺ and intracellular K⁺. A reversal potential could not be found for the current. L-trans-Pyrrolidine-2.4-dicarboxylic acid (PDC), a blocker of Na⁺-dependent glutamate uptake, diminished the glutamate current also in our cells. Application of L-AAA also generated an inward current at negative holding potentials, without a reversal potential, being suppressed if extracellularNa⁺ or intracellular K⁺ was removed. The glutamate uptake blocker, PDC (200 μM), blocked the L-AAA (1 mM) current. Thus, L-AAA proved to be transported by the Na⁺/glutamate transporter of Müller cells. Hence, glutamate currents were diminished by L-AAA competitively with a K_m of 499 μM at a glutamate concentration of 10 μM. The Na⁺/glutamate uptake was less sensitive to DL- and D-AAA block. It is suggested that the blocking effect of AAA on Na⁺-dependent glutamate uptake into glial cells might be involved in the well known glia toxicity of this compound.     

Phagocytic and lysosomal activity in Langerhans cells from sensitized guinea pigs

Guinea pig skins were sensitized and challenged with horseradish peroxidase. Langerhans cell granules contained the horseradish peroxidase. The cytoplasmic locations of horseradish peroxidase in Langerhans cells changed with the passage of time: The antigen was found initially in peripheral Langerhans cell granules and subsequently in those located near the nucleus. The number of lysosome-like dense bodies, or multivesicular bodies increased with time. After 24 hr the antigen was absent from the Langerhans cell granules and seen only in lysosome-like dense bodies, or multivesicular bodies. When acid phosphatase activity was examined, the Langerhans cell granules attached to plasma membranes and rodshaped granules were negative. It was positive in lysosome-like dense bodies, multivesicular bodies, and the bulb part of the Langerhans cell granules. A similar sequence of observations was made when guinea pig skins were sensitized and challenged with ferritin and 2,4-dinitro-1-chlorobenzene. It is concluded that Langerhans cell granules can take up antigens and assume an aspect of the lysosome.     

Effect of maternal starvation on fetal tissue nucleic acid, plasma amino acid and growth hormone concentration in sheep

The measurement of nucleic acids in fetal tissues as well as plasma growth hormone and amino acids was used in conjunction with fractional protein synthetic rates to investigate the mechanism of reduced fetal protein synthesis following acute maternal starvation. The nucleic acid analysis of fetal tissues from fed and 48 h starved ewes (120-130 days gestation) demonstrated a significant reduction in kidney RNA and heart DNA concentration in the starved fetuses. The RNA synthetic capacity (RNA/protein) was also seen to decrease in the starved fetuses both for liver and kidney tissue as was the protein/DNA in the lung tissue. Most revealing, however, were the measurements of RNA and DNA activity or the extent to which the protein synthesizing capacity was realized (g protein/g RNA or DNA/day). Significant reductions were observed in liver and brain RNA activity as well as the DNA activity of liver, lung, kidney and muscle. Plasma aminograms demonstrated reductions in maternal histidine, methionine and isoleucine as well as reductions in fetal glutamate and phenylalanine following starvation. Conversely, the fetal growth hormone levels were seen to rise under the influence of maternal starvation. The impact of maternal nutrient deprivation during gestation on fetal metabolism appears to depend on the ontogenic stage of development of specific tissues at the time the deprivation occurs.     

Horizontal cells isolated from catfish retina contain two types of excitatory amino acid receptors

1. Inward going membrane currents elicited by N-methyl-D-aspartate (NMDA), glutamate (GLU), and glutamate analogues were recorded from isolated catfish (Ictalurus punctatus) cone horizontal cells using the patch-clamp technique in the whole-cell mode. 2. Currents elicited by the N-methyl-D-aspartate receptor agonists NMDA, L-aspartate (ASP) or L-homocysteate (L-HCA) in nominally Mg-free saline were completely blocked by 100 microM 2-amino-5-phosphonovalerate (AP-5) but responses to non-NMDA receptor agonists kainate (KA), quisqualate (QA), or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) in normal Mg2+ saline were unaffected. Responses to GLU were partially blocked. Kynurenic acid (1 mM) effectively blocked responses to all agonists. 3. Concentration-response curves obtained from measured responses in the presence of different NMDA receptor agonists gave Hill coefficients of near 1 indicating a single binding site for channel activation. The rank order of agonist affinity at the NMDA receptor is L-HCA greater than NMDA greater than ASP. Glycine potentiates responses to NMDA in horizontal cells. 4. The NMDA-activated channel is blocked in a voltage-dependent manner by Mg2+, Ni2+, and Co2+ and in a voltage-independent manner by Zn2+. Both the NMDA- and KA-activated channel were permeable to monovalent cations but the NMDA-activated channel appeared to have a greater permeability to Ca2+ than the KA-activated channel. 5. Concentration-response curves measured from responses to the non-NMDA receptor agonists QA, KA, and AMPA gave Hill coefficients of approximately 1.5 suggesting multiple binding sites for channel activation and cooperativity. The rank-order affinity was QA greater than AMPA greater than GLU greater than KA. KA was the most efficacious of the agonists resulting in the largest Imax. Rapid desensitization was observed only in the presence of QA, AMPA, or GLU and this desensitization could be removed by pretreatment with conconavalin A (Con A). 6. Single-channel conductance and mean open time were measured from the fluctuations in current noise in the presence of the agonists. The single-channel conductance estimated from the slope of a linear regression obtained from a plot of the variance of the conductance versus the whole-cell conductance measured in NMDA and ASP was 4.7 pS. The mean channel open time was 1.3 ms. These same parameters measured for KA and QA were 5.7 and 5.9 pS and 1.1 to 1.3 ms, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of bile salts on amino acid transport by rabbit intestine.

The effect of bile salts on alanine absorption across four regional sites of rabbit intestine was examined using an in vivo single-pass perfusion technique. Na-deoxycholate at a concentration of 3 mM reduced alanine absorption across all levels of the intestine, and a higher concentration (10 mM) of Na-taurodeoxycholate (TDC) caused only a minimal reduction of alanine absorption in the jejunum. TDC, however, was more effective in in vitro experiments, causing an incrase in transmural serosal-to-mucosal flux of alanine and phenylalanine, particularly when present in both the mucosal and serosal media. It also reduced the mucosal-to-serosal alanine flux rate when present only in the mucosal medium. The influx of these amino acids across the mucosal brush border membrane was also decreased by TDC. These amino acid transport changes correlated fairly well with some observed histological changes of the intestinal epithelium. This suggests that bile salt inhibition of amino acid absorption is nonspecific in type and can be mainly explained as being the result of an injurious action of these surface-active agents on the rabbit intestine.     

Aberrant amino acid transport in fibroblasts from children with autism

Autism is a developmental, cognitive disorder clinically characterized by impaired social interaction, communication and restricted behaviours. The present study was designed to explore whether an abnormality in transport of tyrosine and/or alanine is present in children with autism. Skin biopsies were obtained from 11 children with autism (9 boys and 2 girls) fulfilling the DSM-IV diagnostic criteria for autistic disorder and 11 healthy male control children. Transport of amino acids tyrosine and alanine across the cell membrane of cultured fibroblasts was studied by the cluster tray method. The maximal transport capacity, V_max and the affinity constant of the amino acid binding sites, K_m, were determined. Significantly increased V_max for alanine (p = 0.014) and increased K_m for tyrosine (p = 0.007) were found in children with autism. The increased transport capacity of alanine across the cell membrane and decreased affinity for transport sites of tyrosine indicates the involvement of two major amino acid transport systems (L- and A-system) in children with autism. This may influence the transport of several other amino acids across the blood–brain-barrier. The significance of the findings has to be further explored.