Inhibitory effect of sugars on the spontaneous lysis of EAC1-8.
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Amino Acids↗
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Basolateral transport of taurine in epithelial cells of isolated, perfused Mytilus californianus gills.
We found that the basolateral surface of the gill epithelium of the marine mussel Mytilus californianus possesses a carrier-mediated process capable of concentrating taurine within epithelial cells. We used retrograde perfusion of gill sections to demonstrate the kinetics, specificity and ion-dependence of taurine transport. [3H]taurine was concentrated relative to a space marker ([14C]mannitol); this accumulation was blocked by the inclusion of 10 mmol l-1 unlabeled taurine in the perfusate. The drop in [3H]taurine uptake at increasing concentrations of unlabeled taurine was fitted to Michaelis-Menten kinetics and indicated a basolateral process with a taurine concentration at which transport is half-maximal (Kt) of 35.3 mumol l-1 and a maximal flux (Jmax) of 0.35 mumol g-1 wet mass h-1. Taurine accumulation on the apical surface had a higher affinity (Kt = 9.5 mumol l-1) and a higher maximum rate of transport (Jmax = 1.23 mumol g-1 h-1). Basolateral transport was inhibited by inclusion in the perfusate of 1 mmol l-1 of another beta-amino acid (beta-alanine), but not by inclusion of alpha-alanine, glutamic acid or betaine. The dependence of basolateral taurine transport on Na+ (when replaced with N-methyl-D-glucamine) was sigmoidal with an apparent Hill coefficient of 2.3, indicating that more than one Na+ is necessary for the transport of each taurine molecule. Complete substitution of Cl- in bathing media reduced taurine accumulation by 90% and 70% on the apical and basolateral surfaces, respectively. Taurine accumulation on both surfaces was reduced by only 20% when Cl- was reduced from 496 to 73 mmol l-1, suggesting that taurine uptake is not significantly influenced by the changes in Cl- concentration accompanying the salinity fluctuations normally encountered by mussels. We estimate that the various Na+ and Cl- gradients naturally encountered by epithelial cells are capable of providing ample energy to maintain a high intracellular concentration of taurine. We suggest that the ability of epithelial cells to accumulate taurine across the basolateral surface from the hemolymph plays a significant role in the intracellular regulation of this important osmolyte and may effect osmolality-dependent changes in the intracellular concentration of taurine.
Amino Acids↗
Effect of taurine on calcium binding to microsomes isolated from rat cerebral cortex.
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Adenosine Triphosphate↗
Regulation of glycogen synthesis and glycolysis by insulin, pH and cell volume. Interactions between swelling and alkalinization in mediating the effects of insulin.
The effects of changes in cell volume and pH on glycogen synthesis and glycolysis and their control by insulin were investigated in hepatocyte cultures. 1. Cell acidification, by increasing [CO2] from 2.5% to 5%, inhibited glycolysis and stimulated glycogen synthesis. The inhibition of glycolysis was also observed in Na(+)-free media and when K+ uptake was inhibited, but the stimulation of glycogen synthesis was abolished under these conditions, suggesting that it is secondary to ionic or volume changes. Alkalinization had converse effects on glycolysis and glycogen synthesis. 2. In HCO3(-)-containing media, replacement of NaCl with sodium acetate or potassium acetate, like acidification with CO2, inhibited glycolysis and stimulated glycogen synthesis. The latter correlated with an increase in cation content. Amiloride, an inhibitor of Na+/H+ exchange, inhibited both the increase in cation content and the stimulation of glycogen synthesis, suggesting that the latter is secondary to cell swelling. 3. Hypo-osmotic swelling increased glycogen synthesis in HCO3(-)-containing media, in both the absence and the presence of Na+ and at both 2.5% and 5% CO2, but it increased glycolysis in the presence of Na+ and at 2.5%, but not at 5%, CO2. In HCO3(-)-free media, during acidification and swelling, glycogen synthesis correlated with pH and not with cell volume, indicating that inhibition by acidification over-rides stimulation by swelling. 4. Stimulation of glycolysis by insulin was not additive with stimulation by alkalinization. The stimulation of glycogen synthesis by insulin was partially suppressed under alkaline conditions; it was markedly suppressed in isosmolar Na(+)-free media and restored by hypo-osmotic swelling. In hypo-osmolar Na(+)-free media insulin prevented the decrease in glycogen synthesis with decreasing [HCO3-], suggesting that it counteracts inhibition by acidification. 5. It is concluded that glycogen synthesis and glycolysis are both stimulated by cell swelling and inhibited by acidification, under certain conditions, but glycolysis is more sensitive to inhibition by acidification and glycogen synthesis to stimulation by swelling. Consequently, simultaneous swelling and acidification is associated with inhibition of glycolysis and stimulation of glycogen synthesis. Stimuli that cause swelling and alkalinization activate both glycogen synthesis and glycolysis, alkalinization being more important in control of glycolysis and swelling in control of glycogen synthesis. Both cell swelling and alkalinization are components of the mechanism by which insulin controls glycogen synthesis and glycolysis.
Acetates↗
The asymmetric orientation of cytochrome b561 in bovine chromaffin granule membranes.
The topological arrangement of cytochrome b561 in the bovine adrenal medullary chromaffin granule membrane was investigated by radiolabeling and immunoprecipitation techniques using antibody raised against the purified cytochrome. The first labeling procedure involved a membrane-permeable amino group labeling reagent, ethyl acetimidate, and two membrane-nonpermeable amino group labeling reagents, isethionyl acetimidate and trinitrobenzenesulfonic acid. The second radiolabeling procedure involved lactoperoxidase-catalyzed iodination of the exposed tyrosines on the membrane-bound proteins. The labeled cytochrome b561 was isolated by immunoprecipitating detergent extracts of treated membranes, followed by electrophoresis of the precipitated cytochrome in polyacrylamide-dodecyl sulfate. From the analysis of both labeling techniques, cytochrome b561 appeared to be a transmembrane protein and a major portion of this protein was cytoplasmically exposed.
Adrenal Medulla↗
Plasma membrane aminophospholipid distribution in transformed murine fibroblasts.
The possibility that the asymmetric distribution of aminophospholipids may be an intrinsic property of mammalian plasma membranes was examined in LM cells, a transformed murine fibroblast cell line. The cells were grown in suspension culture in a chemically defined medium without lipid, protein, or serum and then treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). A maximum of 4% of LM cell plasma membrane phosphatidylethanolamine and 5% of the phosphatidylserine was labelled with TNBS. Furthermore, long chain and unsaturated fatty acids were preferentially esterified to the non-derivatized phosphatidylethanolamine (inner monolayer) as compared to phosphatidylethanolamine derivatized with TNBS (outer monolayer). Isethionyl acetimidate, an alternative non-penetrating reagent, confirmed the results obtained with TNBS and provided supportive evidence for the highly asymmetric distribution of phosphatidylethanolamine; 6% of the phosphatidylethanolamine was labelled with isethionyl acetimidate. When the penetrating reagent methylacetimidate was used, more than 80% of the phosphatidylethanolamine was derivatized. Although the growing of the LM cells in 10% calf serum significantly increased plasma membrane phosphatidylcholine while decreasing phosphatidylethanolamine, calf serum had no significant effect on phosphatidylethanolamine or phosphatidylserine asymmetry.
Animals↗
Chemotactic factor receptors of human PMN leucocytes. I. Effects on migration of labelling plasma membrane determinants with impermeant covalent reagents and inhibition of labelling by chemotactic factors.
The mechanism of stimulation of human PMN leucocyte-directed migration by chemotactic factors was studied by pre-labelling plasma membrane determinants with impermeant covalent reagents and assessing the effects of such modification on spontaneous mitration and chemotaxis in modified Boyden chambers. Pre-treatment of PMN leucocytes with 10(-9) to 10(-6) M isethionyl acetimidate, which selectively labels amino groups, enhanced spontaneous migration and concomitantly inhibited chemotaxis to fragments of the fifth component of complement (C5fr), 12-L-OH-5,8,10,14-eicosatetraenoic acid (HETE) and several formyl--methionyl (f--Met) peptides to an extent that was inversely related to the magnitude of the chemotactic response of untreated PMN leucocytes. Para-chloromercuribenzene sulphonate, which selectively labels sulphydryl groups, inhibited chemotaxis to diverse stimuli without substantially influencing spontaneous migration, while the diazonium salt of sulphanilic acid, which labels several types of plasma membrane determinants, altered neither spontaneous nor chemotactic migration. Incubation of PMN leucocytes with various concentrations of [3H]-isethionyl acetimidate labelled from 33,000 amino groups per PMN leucocyte at 10(-6) M to over 800,000 at 10-4) M, a reaction that was substantially inhibited by chemotactic concentrations of C5fr and HETE, but not by f--Met peptides. Subcellular fractionation of PMN leucocytes labelled with [3H]-isethionyl acetimidate localized the radioactivity to membrane-rich fractions. Free amino groups thus appear to be functionally critical determinants of some chemotactic factor receptors on the plasma membrane of PMN leucocytes.
4-Chloromercuribenzenesulfonate↗
Actions of gamma-aminobutyric acid on sympathetic ganglion cells.
1. Responses of single ganglion cells in the isolated rat superior cervical ganglion to gamma-aminobutyric acid (GABA) applied via the bathing medium were recorded using intracellular micro-electrodes. 2. GABA produced a large fall in cell input resistance, frequently to immeasurable levels. In thirteen cells showing a modest response to 100 muM GABA, input resistance fell from 50-5 +/-9-5 to 15.9 +/- 3-2 Momega (means +/- S.E. of mean). After correction for resistance leaks introduced by the impaling electrode, the resting membrane resistance Rm and the resistance of the GABA-shunt Rg in these cells were calculated to be 79-3 +/- 16-6 and 35-0 +/- 9-5 Momega respectively. 3. Cells with recorded resting membrane potentials greater than -42 mV were depolarized by GABA; at resting potential less than -42 mV they were hyperpolarized...
Aminobutyrates↗
A role for anion transport in the regulation of release from chromaffin granules and exocytosis from cells.
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Binding, Competitive↗
Electrical properties and structure of the frog arachnoid membrane.
We have used an in vitro preparation of the frog arachnoid membrane to study the role of this membrane in the maintenance of the "blood-cerebrospinal fluid (csf) barrier". Electron microscopy showed that the membrane was made up of 10-15 layers of flat epithelial cells joined together by numerous cell junctions. The electrical resistance of the preparation was about 2000 ohms cm2. The steady-state transmural potential difference (pd) ranged up to 45 mV, csf positive, and this eliminated by either the addition of ouabain to the csf, or by replacing the NaC1 with TEA C1. The pd across the membrane increased when bicarbonate was added to the external bathing solutions. We conclude that this pd is due to the active transport of sodium from the subural fluid to the csf. In some preparations the transmural pd was reversed, i.e., csf negative, and this was also abolished by the addition of ouabain to the csf, or by replacing chloride with isethionate. We conclude that this pd is related to active chloride transport. These, and other experiments, lead us to the conclusion that the arachnois membrane is involved in the production of the cerebrospinal fluid and the maintenance of the blood-cerebrospinal fluid barrier.
Animals↗
Contributions of sodium and chloride to ultrastructural damage after dendrotomy.
To determine the contributions of sodium and chloride to ultrastructural changes after mechanical injury, we amputated primary dendrites of cultured mouse spinal neurons in low calcium medium in which sodium chloride had been replaced with either choline chloride or sodium isethionate or sodium propionate. Uninjured cultured neurons were also exposed to the sodium ionophore, monensin. A third set of neurons was injured in medium in which all sodium and calcium chloride had been replaced with sucrose. Neurons injured in low-calcium, low-sodium medium exhibited few ultrastructural changes, except very near the lesion, where there was some dilation of mitochondria and cisternae of the smooth endoplasmic reticulum (SER). Mitochondria in other regions of the neurons developed an electron opaque matrix, and those nearer to the lesion converted to the condensed configuration, characterized by expanded intracristal spaces as well as a dense matrix. If sodium but not chloride was present in the medium, there was some dilation of the Golgi cisternae after injury, as well as some increased electron opacity of the mitochondria. Monensin treated neurons also exhibited dilation of the Golgi cisternae. Neurons injured in sucrose-substituted medium showed none of the changes associated with injury in normal culture medium. These results indicate that sodium influx through the lesion is involved in the dilation of the SER, which is seen even in low-calcium medium, and that a permeant anion, such as chloride, is also involved. This dilation of the SER may result from uptake of calcium released from mitochondria in response to elevated cytosolic sodium. Dilation of the Golgi cisternae appears to be a response only to elevated intracellular sodium. Condensation of the mitochondria after injury is thought to be due to increased demands for ATP synthesis and may involve a "futile cycling" of calcium across the mitochondrial membrane, involving sodium-mediated calcium release in response to elevated intracellular calcium.
Animals↗
Ca2+ modulates an unspecific cation conductance in olfactory cilia of Xenopus laevis.
Olfactory neurones of Xenopus laevis were studied by the patch clamp technique under voltage-clamp conditions. Isolated receptor cells were obtained by dissociating the olfactory mucosa in a Ca(2+)-free solution. Usually some of the resulting isolated olfactory cells lost all of their cilia during the dissociation procedure. Comparing the currents of cells with cilia to those of cells without cilia, a marked difference was found. When all known voltage-gated currents except the Ca(2+)-current were blocked, cells without cilia showed the voltage-gated Ca(2+)-current alone whereas cells with cilia clearly had an additional conductance gc. It could be activated in two ways, either by Ca2+ entry through Ca(2+)-channels or by Ca2+ entry through the Na/Ca-exchanger working in the reversed mode at positive membrane potentials. This ciliar conductance gc had its reversal potential at 0 mV. Replacing extracellular Cl- by isethionate on the one hand, and Na+ by Cs+ or N-methyl-D-glucamine on the other showed that gc was permeable for cations but not for Cl-. In conclusion, there appears to be a Ca(2+)-dependent unselective cation conductance on the cilia of olfactory neurones. The probable role of gc as the last step an IP3/Ca mediated transduction pathway is suggested.
Animals↗
Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. II. Impedance measurements.
In the preceding publication we have reported that, contrary to the prevailing opinion in the literature, the tight-junction tightness of Necturus gall bladder epithelium is not up-regulated by cAMP-mediated or by Ca(2+)-mediated stimulation. This conclusion was based on our observation that the stimulant-induced increase in transepithelial resistance (Rt) occurred only when the lateral intercellular spaces were allowed to collapse, which suggested that the increase reflected primarily or exclusively the increasing resistance of the lateral spaces (Rlis) rather than the postulated increase in tight-junction resistance (Rj). An alternative explanation could have been that the constancy of Rt after space dilatation reflected an increase Rj that was masked by a concomitant fall in apical and basolateral cell membrane resistances Ra and Rbl. To decide between those possibilities we have performed impedance measurements with transepithelial and intracellular microelectrodes on Necturus gall bladder epithelium. Applying previously developed analysis procedures, the measurements readily showed that elevation of intracellular Ca2+ concentration increased Rlis, but left Rj as well as Ra and Rbl quasi constant. Experiments with forskolin, theophylline or isobutylxanthine, on the other hand, were less clear. These stimulants activated an apical Cl- conductance, which drastically reduced Ra and apparently caused low-frequency polarization effects that could not be accounted for by the classical epithelial equivalent circuit. After elimination of the polarization phenomena by uni- or bilateral substitution of Cl- by isethionate or sulphate, however, we were able to demonstrate that Rj remains constant under cAMP-mediated stimulation irrespective of whether the lateral spaces are kept open or are allowed to collapse. We conclude that the tight-junction resistance of Necturus gall bladder epithelium is not controlled by intracellular Ca2+ or by cAMP-mediated stimulation.
Animals↗
Further evidence for an inverse relationship between macula densa NaCl concentration and filtration rate.
It has been concluded that tubulo-glomerular feedback mechanism is triggered by changes in NaCl concentration ([NaCl]) at the macula densa. This conclusion is based on the demonstration that changes in filtration rate produced during retrograde perfusion of the loop of Henle depend upon the perfusate [NaCl]. Experiments were performed to evaluate whether the effect on glomerular function of orthograde perfusion of the loop of Henle is consistent with this conclusion. Early proximal flow rate (VEP), stop-flow pressure (PSF), early distal chloride concentration ([C]), and flow rate were measured during perfusion of the loop of Henle with mannitol solution (300 mosm kg-1), 30mM NaCl + mannitol (300 mosm kg-1), 140 mM Na isethionate and artificial tubular fluid. When distal flow exceeded 10 nl min-1, the magnitude of the glomerular response was predictable from the [Cl]. The linear regression line, delta VEP = -0.27 [Cl] + 4.3, did not differ from that obtained previously with the retrograde technique. Retrograde perfusion with 140 mM Na isethionate was without effect on VEP. We conclude that the effect on glomerular function of perfusion of the loop of Henle in either an orthograde or a retrograde direction with these solutions depends upon the chloride concentration at the macula densa.
Animals↗
Effects of chemical modification of amino groups by two different imidoesters on voltage-clamped nerve fibres of the frog.
Voltage clamped single nerve fibres of the frog Rana esculenta were treated with the amino groups specific reagents ethyl acetimidate and isethionyl acetimidate. Ethyl acetimidate is lipid soluble, relatively non-polar and can penetrate a membrane. Isethionyl acetimidate is lipid-insoluble, polar and membrane-impermeant. Treatment with ethyl acetimidate caused an irreversible reduction of Na currents and a shift of the voltage dependence of the steady-state sodium inactivation, h infinity (E), in the hyperpolarizing direction. The voltage dependence of sodium activation was much less affected and a small shift into the depolarizing direction was observed. By contrast, the non-permeant reagent did not reduce the sodium currents and the shifts of the h infinity (E) curve were smaller than the shifts caused by ethyl acetimidate. Furthermore, a small shift of the voltage-dependence of activation in the hyperpolarizing direction was observed. As the modification procedure with imidoesters does not cause a change of net charge, the results cannot be explained by an effect of surface charge modification; rather, they seem to be due to a direct effect of amino group modification on the voltage dependence of inactivation.
Alkanesulfonates↗
The influence of chloride on the ouabain-sensitive membrane potential and conductance of crayfish giant axons.
1. Resting potential and current-voltage relation were measured in crayfish giant axons bathed in chloride-free and sodium-free solutions with and without ouabain. 2. Chloride-free solution caused a transient depolarization but did not alter the steady-state membrane potential. Utilizing isethionate as an anion substitute, the membrane resistance increased 12.5%. 3. In the absence of extracellular chloride, ouabain (0.5-1 mM) depolarized the axon 6-7 mV. The shape of the current-voltage relation did not change but the curve was shifted along the current axis. 4. These results indicate that ouabain inhibits a steady-state hyperpolarizing electrogenic pump current of approximately 3 muA/cm2. 5. Extracellular sodium removal from axons equilibrated in chloride-free solutions transiently hyperpolarized the membrane 6-7 mV without a change in membrane resistance. The transient hyperpolarization was ouabain and temperature sensitive. The steady-state potential reached in sodium-free and chloride-free solution was not ouabain sensitive. Temperature sensitivity of the steady-state membrane potential was greatly reduced. 6. The transient hyperpolarization produced by extracellular sodium removal was metabolically driven and may present the expression of a sodium efflux transport current of 7.0-7.5 muA/cm2. 7. Using electrophysiologically measured parameters, sodium and potassium conductance, influx and efflux currents and the coupling ratio for sodium/potassium transport are calculated from a modification of the conductance equation. 8. The sodium/potassium transport coupling ratio for steady-state conditions was estimated at 5:3 (1.67:1).
Animals↗
The stimulus-secretion coupling of glucose-induced insulin release. XLV. The anion-osmotic hypothesis for exocytosis.
Replacement of extracellular chloride by the impermeant anion isethionate, increase in extracellular osmotic strength by addition of sucrose, or exposure to the anion transport blocking agent probenecid inhibited insulin release evoked by glucose or alpha-ketoisocaproate in rat isolated islets. The inhibition of insulin release due to Cl- substitution was associated with a modest decrease in glucose oxidation, but no significant change in glucose-stimulated 45Ca net uptake by the islets. In the isolated perfused rat pancreas, the isethionate- or sucrose-induced inhibition of insulin release was a rapid and rapidly reversible phenomenon. Chloride substitution by isethionate inhibited more severely the second than the first phase of the secretory response to glucose, and failed to affect the insulin response to gliclazide. A chemosmotic mechanism for exocytosis, as proposed for epinephrine and parathyroid hormone release, may also be involved in insulin release.
Alkanesulfonates↗
Isethionate and taurine transport sites at brain cell membranes: influx studies with brain slices.
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Alkanesulfonates↗