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PubMed1977

Correlation of structure and active transport in the teleost nephron.

In the present study we have extended our investigations concerning the correlation between ultrastructure and active transport in the isolated flounder nephron. The composition of the fish nephron is defined in ultrastructural terms and its behavior when incubated in vitro under short term and long term culture conditions is described. Using the in vitro system originally described by Forster, a variety of inhibitors and conditions which modify cell structure and function were tested. Ultrastructure was correlated with chlorphenol red dye transport. In general, conditions altering active transport also markedly altered cellular ultrastructure. The principal alterations consisted of membrane changes involving various organelles--most importantly the plasma membrane and the mitochondria. Conditions associated with irreversible cell injury could be rapidly produced by interference either with mitochondrial ATP synthesis or with the integrity of the plasma membrane. Both of these rapidly lead to irreversible events which are preceded by reversible structural changes. Organelle changes progress in a rather well-defined sequence of reversible and irreversible stages which are defined. One difference between the two types of interactions is the presence of intramitochondrial calcification which does not occur with direct modification of the mitochondrial electron transport system. The concept of utilizing long term explant organ cultures of fish nephrons for environmental studies is introduced.

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PubMed1979

High-affinity uptake of gamma-aminobutyric acid in cultured glial and neuronal cells.

Both glial and neuronal cells maintained in primary culture were found to accumulate [3H]GABA by an efficient "high-affinity" uptake system (apparent Km = 9 muM, Vmax = 0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1mM) also strongly inhibited uptake of [3H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, beta-alanine, and 2,4-diaminobutyrate) inhibited uptake of [3H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine, L-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (Km = 92 muM, Vmax = 0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [3H]GABA against a concentration gradient. Apparent Km of this uptake was relatively high (819 muM), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, non of the nontransformed continuous cell lines of either tumoral (glioma, C6; neuroblastoma, M1; M1NN) or normal (NN;I6) origin actively accumulated [3H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.

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PubMed1976

Transitory postnatal hemolysis of calf red cells by amino acids.

Among the amino acids which can be solubilized to give a concentration of 300 mm at near physiological pH, histidine and proline caused a complete hemolysis of newborn calf but not of adult cow red cells within 20 to 30 minutes at 38 degrees C. While hydroxyproline, valine, and serine resulted in a partial lysis of calf cells, threonine, glutamine, and glycine were ineffective. In this communication, emphasis has been focused on the mode of the lytic process by histidine which was found to be affected by several governing parameters including the pH, temperature and the extracellular salts in the solution. Unlike human red cells suspended in isotonic histidine, both calf and cow cells lost little Na and K ions. In the presence of 300 mm histidine, both calf and cow cells displayed an instantaneous uptake of histidine amounting to 20 to 45 mumoles/ml RBC followed by a slow influx rate of 0.25 to 0.5 mumoles/ml RBC X min. The extent to which histidine entry was allowed by the cell was counterbalanced by Cl- efflux, resulting in little change in cell volume prior to hemolysis. Moreover, histidine-induced hemolysis can be prevented by 1 mm or lower PCMBS without a discernible effect on histidine influx suggesting a possible membrane lesion or damage at the outer surface of the cell. Hemolysis induced by histidine decreased substantially when a calf reached two months of age at which time the red cells containing the fetal hemoglobin are virtually depleted. The results of hemoglobin electrophoresis obtained during this postnatal period revealed that those cells resistant to histidine hemolysis almost invariably contain the adult type hemoglobin suggesting a selective, specific action of the amino acids on the fetal cells.

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PubMed1979

Studies on lithium transport across the red cell membrane. V. On the nature of the Na+-dependent Li+ countertransport system of mammalian erythrocytes.

Ouabain-resistant Na+-Li+ countertransport was studied on erythrocytes of man, sheep, rabbit, and beef. A transport system, exchanging Li+ for Na+ in a ratio of 1:1, was present in all four species. Li+ uptake by the exchange system increased 30-fold in the order man less than HK-sheep less than LK-sheep less than rabbit less than LK-beef. This order is identical to that of ouabain-resistant Na+-Na+ exchange in these species, but bears no relation to the Na+-K+ pump activity. The activity of the Na+-Li+ exchange system varied up to 7 and 16-fold among individual red cell specimens from man and beef, the variability being much smaller in sheep and rabbit erythrocytes. The affinities of the system for Li+ and Na+ were similar among the species and individuals (half saturation of the external site at about 1 mM Li+ and 50 mM Na+, respectively). 50-60% of Na+-Li+ exchange was blocked by N-ethylmaleimide in all species. p-Chloromercuribenzene sulfonate inhibited the exchange only in beef and sheep erythrocytes (60-80%). The two SH-reagents act by decreasing the maximum activity of the system, whilst leaving its affinity for Li+ unaltered. Phloretin was a potent inhibitor in all species. 1 mM each of furosemide, ethacrynic acid, and quinidine induced only a slight inhibition. The Na+-Li+ exchange of human and beef erythrocytes increased 3.5-fold upon elevation of the extracellular pH from 6 to 8.5, the pH-dependence arising from a change in affinity of the system for the cations and being similar to that reported for ouabain-resistant Na+-Na+ exchange in beef erythrocytes. It is concluded that a transport system exists in the red cell membranes of the four species which can mediate ouabain-resistant exchange of either Na+ for Na+, Na+ for Li+, or Li+ for Li+. The exchange system exhibits essentially identical transport characteristics in the four species, but shows a marked inter- and intra-species variability in maximum transport capacity and some differences in susceptibility towards inhibitors. A similar transport system is probably present also in other tissues. The exchange system seems to be distinct from the conventional Na+-K+ pump and shows no clear relation to one of the furosemide-sensitive, ouabain-resistant Na+ transport systems described in the literature.

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PubMed1979

Studies on lithium transport across the red cell membrane. VI. Properties of a sulfhydryl group involved in ouabain-resistant Na+-Li+ (and Na+-Na+) exchange in human and bovine erythrocytes.

The reactivity of the SH-group essential for ouabain-resistant Na+-Li+ (and Na+-Na+) exchange and its location within the membrane are studied on human and beef erythrocytes and beef red cell ghosts. N-ethylmaleimide (NEM), 1,6-hexane dimaleimide, and iodoacetamide can induce an irreversible, partial inhibition of Na+-Li+ exchange in erythrocytes of the two species. The development of the inhibition due to the alkylating agents is greatly accelerated by external Na+ and Li+. The inhibition takes 3 min (NEM) and 60 min (iodoacetamide) to come to completion in isotonic Na+ media, but is hardly detectable in choline+, K+ or Mg2+ media. The transport site of the exchange system and the site promoting NEM binding exhibit similar affinities for external Na+. The impermeable, monofunctional glutathione derivative of 1,6-hexane dimaleimide does not inhibit Na+-Li+ exchange. The mercurials PCMBS, PCMB, and Hg2+ inhibit Na+-Li+ exchange in beef, but not in human erythrocytes. The inhibitory action of PCMBS, being slightly accelerated by external Na+, is fully reversed by penetrating thiols such as 2-mercaptoethanol, whilst glutathione, an impermeable thiol, is ineffective. Pretreatment with PCMBS affords partial protection from the irreversible inhibition caused by NEM. Oxidation with copper orthophenanthroline inhibits Na+-Li+ exchange only when performed in the presence of penetrating thiols such as 2-mercaptoethanol. It is concluded that the SH-reagents studied inhibit Na+-Li+ exchange by modifying an essential SH-group of a membrane protein in such a way that the turnover number of the exchange system is reduced. This SH-group is separated from both the red cell exterior and interior by a penetration barrier and seems to be distinct from the cation binding site. The action of external Na+ and Li+ in promoting the reaction of alkylating inhibitors is interpreted to result from a conformational change of the transport protein induced by the binding of external Na+ or Li+.

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PubMed1977

Differences between resting and insulin-stimulated amino acid transport in frog skeletal muscle.

We have compared some features of the resting and the insulin-stimulated uptake of alpha-aminoisobutyrate (AIB) in frog skeletal muscle. We found a substantial difference between the two processes, namely, that resting AIB uptake is Na-independent while the insulin-stimulated fraction of the AIB uptake is Na-dependent. Since the amino acid transport systems in frog skeletal muscle are poorly characterized, we have also surveyed some of their properties. One of the most interesting findings of this survey is that both the uptake and efflux of AIB are inhibited by low concentrations of PCMBS (parachloro-mercury-benzene sulfonic acid 5 X 10(-5) M). In contrast, the carrier mediated transport of basic amino acids is neither inhibited by this mercurial agent nor accelerated by insulin. The action of PCMBS strongly suggests the presence of a critical sulfhydryl group in the amino acid carrier system utilized by AIB. This group is exposed to the outside solution since PCMBS penetrates cell membranes poorly, and in addition its inhibitory actions were reverted by agents that do not penetrate the cell membrane like albumin or glutathione.

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PubMed1975

Mercurial perturbation of brush border membrane permeability in rabbit ileum.

The sulfhydryl reagents Hg++ and p-chloromercuribenzene sulfonate (PCMBS) at millimolar concentrations reduced the mucosal entry of sugars and amino acids to 80-90% of control levels within several minutes. Based on 50% levels of inhibition, Hg++ proved to be 20 and 10 times as potent as PCMBS in blocking sugar and amino acid transport, respectively; both systems were equally sensitive to Hg++. Concomitant measurements of 203Hg-PCMBS demonstrated a progressive tissue uptake, which, unlike inhibition, did not saturate with increasing times of exposure, thus suggesting appreciable epithelial entry with prolonged exposures (less than 30 min at 1 mM). At similar dose levels, no significant change in mucosal Na+ entry was detected. Inhibition was not reversed by 30-min washes in cholinesalt solutions; however, 10-min exposures to dithiothreitol [10 mM] reversed Hg++ and PCMBS inhibition by 40 and 100%, respectively. Alanine and galactose influx kinetics measured at concentrations of 0-100 mM exhibited a linear or diffusional entry component in addition to the usual saturable component for both control and Hg++-treated ileum. The presence of a diffusional term in the flux equation resulted in two sets of parameters giving nearly equal fits to these measurements. It was shown that this ambiguity could be resolved by determining the change in diffusional entry with Hg++ treatment. A 20-min exposure to 0.5 mM Hg++ caused an increase from 0.050 and 0.045 to 0.064 and 0.070 cm/hr in the coefficient of diffusional entry for alanine and galactose, respectively. On the basis of this increase, it is argued that Hg++ causes a decrease in Jmax and little change in Km for both transport mechanisms. This analysis has a general bearing on kinetic measurements of transport in which passive fluxes are comparable to those mediated by specific pathways. The alanine results are consistent with bimolecular reactions between mercurial and two membrane inhibitory sites, each producing approximately 40% reduction in membrane translocation rate. The estimated reaction rate constants were 5.0 and 0.4 mM min.

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PubMedDate not supplied

Trypan Blue as a marker of plasma membrane permeability in alloxan-treated mouse islet cells.

Suspensions of pancreatic islet cells from noninbred ob/ob-mice were incubated with Trypan Blue. Microscope photometry showed that apparently viable cells excluded the dye completely, whereas the nuclei of nonviable cells accumulated Trypan Blue by a saturable process. The nucleus-to-medium dye gradient was more then 30:1 in media containing 0.1% or less Trypan Blue. The apparent affinity constant for nuclear binding of the dye was 3.1 X 10(4)l/mol. Albumin partially inhibited the nuclear staining. More than 0.5% Trypan Blue in the medium was toxic per se. In the absence of albumin, 0.5 or 20 mmol/l alloxan, 1 mmol/l N-ethylmaleimide, or 0.1 mmol/l chloromercuribenzene-p-sulphonic acid, but not 20 mmol/l streptozotocin, increased the frequency of islet cells stained with 0.1% Trypan Blue. The absorbance of nuceli was also increased in cells treated with alloxan or N-ethylmaleimide, but not in those treated with chloromercuribenze-p-sulphonic acid. It is concluded that alloxan rapidly increases the permeability of the plasma membrane in mouse beta-cells. This action of alloxan appears to be more acute than any such effect of streptozotocin.

4-Chloromercuribenzenesulfonate↗
PubMed1976

Purification and properties of a low molecular weight protein factor of mitochondrial energy-linked functions.

1. A soluble protein with a molecular weight of 11-12-10(3) has been isolated from bovine-heart mitochondria, which stimulates the following ATP-dependent reactions of submitochondrial particles treated with 0.6 mM EDTA and 1 M NH4OH: reverse electron transfer from succinate to NAD, transhydrogenation from NADH to NADP, and ATP-Pi exchange. The factor has no effect on the NADH oxidase, succinate oxidase and ATPase activities of the particles. 2. The stimulatory effect of the factor in the ATP-dependent reduction of NAD by succinate is 12 mumol-min-1-mg-1 of the factor protein. However, the NH4OH-EDTA treated particles are saturated for maximal activation of the above reaction by very small amounts of the factor (about 20-40 mug factor per mg particle). 3. Electrophoresis of the factor preparation on polyacrylamide gels showed a single protein band plus a nonprotein material which moved at the dye front and was weakly stained with Coomassie Blue. The protein was shown to be required for activation of the particles; whether the fast-moving, nonprotein material is also required is not known. 4. The factor is inhibited by mercurials and N-ethylmaleimide. The former, but not the latter, inhibition is completely reversed by 1,4-dithiothreitol. 5. The NH4OH-EDTA treated particles are also stimulated by rutamycin up to about 0.1 nmol of rutamycin per mg particle; higher rutamycin concentrations inhibit. Depending on the particle preparation, the factor stimulates up to about 3 nmol per mg particle, but does not inhibit at higher concentrations. In addition, under certain conditions in which appropriate concentrations of rutamycin fail to stimulate the particles, the factor still does.

4-Chloromercuribenzenesulfonate↗
PubMed1975

Requirement of heat and metabolic energy for the expression of inhibitory action of colicin K.

Escherichia coli B, induced for beta-galactoside permease, can accumulate thio-methyl-beta-galactoside in the cell even at 0 degrees D. At this temperature, cells adsorb colicin K but the adsorbed colicin does not inhibit thiomethyl-beta-galactoside uptake. Inhibition by colicin K is, however, seen at 0 degrees C after exposure of the colicin K-cell complex to a high temperature: a greater degree of inhibition occurs with increasing temperature or duration or exposure. There is a transition point at around 21 degrees C in Arrhenius plots of this colicin K activation reaction. If inhibitors of energy yielding reactions are present during the heat treatment, the inhibitory action of colicin K (as measured by thiomethyl-beta-galactoside uptake after returning the colicin K-cell complex to 0 degrees C and removal of the inhibitors) is prevented. These results indicate that adsorbed colicin K is converted into the active state only in the presence of metabolic energy and that cell surface fluidity appears to be concerned in this process.

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PubMed1976

Stimulation of Na+ transport across the toad urinary bladder by p-chloromercuribenzene sulfonate.

The sulfhydryl reagent p-chloromercuribenzene sulfonate increased the ISC across substrate-replete toad urinary bladder when applied to the mucosal (apical) surface. This increase was accounted for by an increased mucosal to serosal net flux of Na+. In the absence of substrate, the rise in ISC was accompanied by an irreversible increase in tissue conductance which was not apparent in the replete preparation. These findings suggest that p-chloromercuribenzene sulfonate may be useful in marking mucosal functions associated with the Na+ transport apparatus.

4-Chloromercuribenzenesulfonate↗
PubMed1976

Involvement of sulfhydryl groups in the action of the insulin and radiation on thymocyte Na+-dependent amino acid transport.

1,p-Chloromercuribenzene sulfonate concentrations less than 10(-5) M stimulate the uptake by thymocytes of 2-aminoisobutyrate, a non-metabolized amino acid. At concentrations greater than 10(-5) M of this reagent, transport is impaired and cell viability is effected. In contrast, 5,5'-dithiobis-(2-nitrobenzoate) between 10(-4) and 10(-6) M produces only stimulation of 2-aminoisobutyrate uptake after treating for 10 min. 2. Treatment of thymocytes with 10(-4)M 5,5'-dithiobis-(2-nitrobenzoate) reveals at least three categories of reactive SH groups. Titration of the most rapidly reacting category, 4 - 10(7)-7 - 10(7)/cell, activates 2-aminoisobutyrate transport to the same extent as does p-chloromercuribenzene sulfonate. Cells treated with 10(-6) M insulin showed a 30-50% reduction in the number of sulfhydryl groups that could be titrated with 5,5'-dithiobis-(2-nitrobenzoate). In thymocytes treated with 10(-6) M p-chloro(203Hg)mercuribenzene sulfonate, addition of 10(-6) or 10(-9) M insulin before treatment with the sulhydryl reagent again reduces the number of titrable SH groups by 20%. 3. Insulin (10(-10)-10(-6) M) also stimulates 2-aminoisobutyrate uptake, but the effects of insulin and SH blocker are not additive. 4. Insulin, but not p-chloromercuribenzene sulfonate, prevents the impairment of 2-aminoisobutyrate transport caused by gamma-irradiation. Treatment of cells with p-chloromercuribenzene sulfonate prior to irradiation increases the radiation impairment of 2-aminoisobutyrate transport. 5. gamma-irradiation reduces the number of 5,5'-dithiobis-(2-nitrobenzoate) reactive sulfhydryl residues by 37%. 6. A model for the action of insulin and irradiation on 2-aminoisobutyrate transport is presented.

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