Chemical modification of the Na+/H+ exchanger of thymic lymphocytes. Inhibition by N-ethylmaleimide

A Na+/H+ exchanger is involved in the regulation of cytoplasmic pH and cellular volume in a variety of cells. Little is known about the molecular nature of this exchanger. The purpose of this study was to survey a variety of group-specific covalent reagents as potential inhibitors of the exchanger. Na+/H+ countertransport activity was assayed as the amiloride-sensitive rate of Na+-induced alkalinization in acid-loaded lymphocytes, or as the rate of swelling in cells suspended in sodium propionate medium. Activity was not affected by proteinases or by carboxyl-group and amino-group specific reagents. A significant inhibition was produced by diethylpyrocarbonate, a histidine-specific reagent and by N-ethylmaleimide, a sulfhydryl group reagent. A similarly reactive but nonpermeating sulfhydryl agent, glutathione-maleimide, failed to inhibit Na+-H+ exchange. Moreover, the reaction with N-ethylmaleimide was sensitive to changes in the cytoplasmic pH. The data suggest that the chemically reactive groups of the Na+/H+ exchanger of lymphocytes have limited exposure to the extracellular medium but that an internally located sulfhydryl group is critical for the cation-exchange activity.     

Asymmetry of the Na+/H+ antiport of dog red cell ghosts. Sidedness of inhibition by amiloride

Amiloride is a potent inhibitor of the Na+/H+ antiport. Inhibition is generally competitive with extracellular Na+ and therefore believed to result from binding to the outward-facing transport site. It is not known whether amiloride can interact with the internal aspect of the antiport. This question was addressed by trapping the drug inside resealed dog red cell ghosts. The antiport, which is quiescent in resting ghosts, was activated by acid-loading the cytoplasm. This was accomplished by exchanging extracellular Cl- for internal HCO-3 through capnophorin, the endogenous anion exchanger. The activity of the Na+/H+ antiport was detected as an increase in cell volume, resulting from the net osmotic gain associated with coupled Na+/H+ and Cl-/HCO-3 exchange, or as the uptake of 22Na+. Intracellular amiloride, at concentrations in excess of 100 microM, failed to inhibit Na+/H+ exchange. This is approximately 10 times higher than the concentration required for half-maximal inhibition when amiloride is added externally. Independent experiments demonstrated that failure of internal amiloride to inhibit exchange was not due to leakage of the inhibitor, to differences in pH, or to binding or inactivation of amiloride by the soluble contents. It was concluded that the antiport is functionally asymmetric with respect to amiloride. This implies that the transport site undergoes a conformational change upon translocation across the membrane or, alternatively, that a second site required for amiloride binding is only accessible from the outside.     

Transmembrane signaling by the B subunit of cholera toxin: increased cytoplasmic free calcium in rat lymphocytes

It has previously been shown that the B subunit of cholera toxin, which binds solely to the plasma membrane ganglioside GM1, stimulates the proliferation of rat thymic lymphocytes (Spiegel, S., P. H. Fishman, and R. J. Weber, 1985, Science [Wash. DC], 230:1285-1287). The purpose of this study was to identify which transmembrane signaling system(s) are activated by the B subunit of cholera toxin. We compared the effects of B subunit and concanavalin A (Con A), a potent mitogenic lectin, on a number of second messenger systems that are putative mediators of T cell activation. Changes in the fluorescence of quin2-loaded cells revealed that mitogenic doses of either B subunit or Con A induced rapid and sustained increases in cytoplasmic free Ca2+ ([Ca2+]i). Within 5 min, [Ca2+]i increased from a basal level of 69 +/- 4 to 136 +/- 17 and 185 +/- 24 nM, respectively. The effects of B subunit and Con A were additive and largely dependent on the presence of extracellular Ca2+, though release of Ca2+ from intracellular stores could be detected for Con A, but not B subunit, using indo-1. The B subunit had no effect on either inositol phosphate levels or on the distribution of protein kinase C, indicating that, unlike Con A, the B subunit does not activate phosphoinositide hydrolysis. Fluorimetric measurements on cells loaded with bis(carboxyethyl)-5,6-carboxyfluorescein revealed that Con A induced a rapid cytoplasmic alkalinization via activation of Na+/H+ exchange, whereas B subunit had no effect on intracellular pH. Finally, by monitoring bis-oxonol fluorescence, we found that Con A induced a small hyperpolarization of the membrane potential, whereas B subunit had no acute effect. These data suggest that the biological effects of B subunit are mediated by an increase in [Ca2+]i resulting from a net influx of extracellular Ca2+.     

Actin assembly in electropermeabilized neutrophils: role of G-proteins

Polymerization of microfilaments, one of the responses triggered in neutrophils by stimuli such as the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), involves the conversion of actin from the monomeric to the filamentous form. The exact sequence of events responsible for this conversion remains to be defined, but its susceptibility to inhibition by pertussis toxin provides indirect evidence that GTP-binding proteins (G-proteins) are involved. In this report, electropermeabilized cells were used to obtain more direct evidence of a role for G-proteins in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin and flow cytometry were used to monitor the formation of filamentous actin. GTP-gamma-S, a nonhydrolyzable analogue of GTP and aluminum fluoride, which in combination with GDP can activate G-proteins, stimulated actin assembly in electropermeabilized cells but had only marginal effects on intact cells. fMLP-induced actin polymerization in permeabilized cells was inhibited by pretreatment with GDP-beta-S, an analogue of GDP that stabilizes the inactive form of G-proteins. In contrast, stimulation by phorbol 12-myristate 13-acetate (PMA) was largely unaffected by GDP-3-S. These observations indicate that activation of G-proteins is essential for actin assembly induced by receptor-dependent stimuli such as fMLP. Moreover, GTP-binding proteins do not seem to be required in the late stages of the signalling cascade, i.e. after stimulation of protein kinase C.     

Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytosolic acidification in human T lymphocytes

We have investigated the patterns of cytosolic pH and Ca2+ ([Ca2+]i) changes after exposure of human peripheral blood T cells to different mitogenic and non-mitogenic ligands. Using ligands that have different accessory cell requirements and varying effect on [Ca2+]i or cell proliferation, we observed that intracellular acidification occurred only with agents that increased [Ca2+]i. However, treatment of the cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in significant cytosolic alkalinization without detectable acidification, but did not affect the proliferative responses to mitogenic ligands and was a potent co-mitogen with non-mitogenic ligands. These data indicate that initial acidification or alkalinization responses are not essential for early activation or triggering of DNA synthesis.     

Effect of glucose on induction of delta-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead

In the present work we have been able to demonstrate that phenobarbital and lead exert an inducing effect on the biosynthesis of delta-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the induction effect produced by phenobarbital in this in vitro system. Glucose inhibits the induction of delta-aminolevulinic acid synthase and ferrochelatase. This repression effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. No glucose effect was observed on the phenobarbital- and lead-mediated inductions of cytochrome P-450. he present results add more experimental evidence to support the concept that the last enzyme of the heme pathway is inducible, and as such may have a significant role in regulatory mechanisms of porphyrin and heme biosynthesis.     

Activation of Na+/H+ exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification

After swelling in hypotonic solutions, peripheral blood mononuclear cells (PBM) shrink toward their original volumes. Upon restoration of isotonicity, the cells initially shrink but then regain near-normal size again. This regulatory volume increase (RVI) is abolished by removal of Na+o or Cl-o or by addition of amiloride. RVI is unaffected by removal of K+o or by ouabain and is only partially inhibited by 1 mM furosemide. As a result of increased influx, the cells gain both Na+ and K+ during reswelling. In contrast, only Na+ content increases in the presence of ouabain. Amiloride largely eliminates the changes in the content of both cations. Using diS-C3-(5), no significant membrane potential changes were detected during RVI, which suggests that the fluxes are electroneutral. The cytoplasmic pH of volume-static cells was measured with 5,6-dicarboxyfluorescein. After acid loading, the addition of extracellular Na+ induced an amiloride-inhibitable alkalinization, which is consistent with Na+/H+ exchange. Cytoplasmic pH was not affected by cell shrinkage itself, but an internal alkalinization, which was also amiloride sensitive and Na+ dependent, developed during reswelling. In isotonic lightly buffered solutions without HCO-3, an amiloride-sensitive acidification of the medium was measurable when Na+ was added to shrunken PBM. K+ was unable to mimic this effect. The observations are compatible with the model proposed by Cala (J. Gen. Physiol. 1980. 76:683-708), whereby an electroneutral Na+o/H+i exchange is activated by osmotic shrinking. Cellular volume gain occurs as Cl-o simultaneously exchanges for either HCO-3i or OH-i. Na+i is secondarily replaced by K+ through the pump, but this step is not essential for RVI.