Cation transport in oxidant-stressed human erythrocytes: heightened N-ethylmaleimide activation of passive K+ influx after mild peroxidation.

Normal and chronically dehydrated (hereditary xerocytosis) human red cells were subjected to mild peroxidative treatment (315 microM hydrogen peroxide (H2O2), 15 min) in the presence of azide. The subsequent expression of passive (ouabain-resistant) K+ transport activities was analyzed by measurement of 86Rb+ influx. Peroxidation of normal red cells did not affect basal K+ transport activity, but the increment in K+ influx elicited by 0.5 mM N-ethylmaleimide (NEM) was increased 3-fold. The enhanced K+ influx was chloride-dependent, but only partially inhibited by 0.1 mM furosemide. Stimulated activity declined progressively after NEM activation, but could be restored by a second NEM treatment. Prior conversion of hemoglobin to the carbonmonoxy form abolished the response to peroxide, while 200 microM butylated hydroxytoluene (BHT) exerted only partial inhibition, suggesting that the effect of H2O2 requires interaction of activated, unstable hemoglobin species with the membrane, but that lipid peroxidation is not sufficient. Peroxidation following NEM treatment also enhanced NEM activation, indicating that enhancement does not require altered NEM reactions with stimulatory or inhibitory sites. Passive K+ transport in hereditary xerocytosis red cells was not activated by NEM, with or without H2O2 pretreatment. The results demonstrate that modest peroxidative damage to red cells can heighten the activation of a transport system that is thought to be capable of mediating net K+ efflux and volume reduction in cells that express it. Models are proposed in which the effects of NEM, H2O2, cell swelling and other factors are mediated by conformational changes in a postulated subpopulation of anion channel (Band 3) molecules that bind the K+ transporter.

The role of membrane protein sulfhydryl groups in hydrogen peroxide-mediated membrane damage in human erythrocytes.

The formation of spectrin-hemoglobin complex following treatment of red cells with hydrogen peroxide (H2O2) has previously been shown to be associated with alterations in cell shape, decreased membrane deformability and increased recognition of modified cells by anti-IgM immunoglobulin in a phagocytic assay by monocytes. Prior treatment with carbon monoxide completely inhibited the H2O2-associated membrane changes, indicating a role for oxidized hemoglobin in the complex formation. Also, in a cell-free system, blockage of sulfhydryl (SH) groups on purified spectrin by N-ethylmaleimide significantly reduced the complex formation, suggesting a role for SH groups of spectrin in crosslinking process. The present study was undertaken to examine the role of SH blockade by N-ethylmaleimide on intact red cells undergoing oxidative damage. Pretreatment of erythrocytes with N-ethylmaleimide at concentrations ranging from 0.1 to 0.2 mM resulted in decreased lipid peroxidation and spectrin hemoglobin crosslinking. Moreover, pretreatment with N-ethylmaleimide resulted in less marked alterations in cell shape and membrane deformability as well as reduced recognition of peroxidized cells by antiglobulin serum. N-Ethylmaleimide treatment had no effect on methemoglobin formation. Studies with 14C-labeled N-ethylmaleimide showed that over 50% of N-ethylmaleimide was incorporated into spectrin. Pretreatment of cells with higher concentrations of N-ethylmaleimide (over 0.2 mM) was associated with membrane dysfunction independent of H2O2. These results imply that blocking of reactive SH groups leads to reduced interaction of spectrin with oxidized globin. These data, along with our prior observations, indicate that SH groups on spectrin play an important role in hemoglobin oxidation-induced formation of spectrin-hemoglobin complex and the resultant deleterious effects on membrane properties.

Ileal mucosal cyclic AMP and Cl secretion: serosal vs. mucosal addition of cholera toxin.

Changes in ion transport and cyclic AMP (cAMP) concentration produced by addition of cholera toxin to the serosal side of isolated rabbit ileal mucosa (CTs) were compared to the changes produced by addition to the mucosal side (CTm). CTs increased short-circuit current (SCC) as did CTm but it did so more slowly. CTs, unlike CTm, did not significantly decrease electrical conductance. Inhibition of the SCC response to theophylline, a measure of preexisting secretion, was almost complete 180 min after CTm but was not yet significant 180 min after CTs. Longer (280 min) after CTs, the SCC response to theophylline was reduced by 59%, a significant reduction but less than that caused by CTm. A statistically significant change in net Cl flux could not be demonstrated after CTs, although at 280 min the measured flux was halfway between the fluxes for control and CTm tissues. Cyclic AMP concentrations were determined at 190 min, 10 min after addition of theophylline. CTs, despite little or no effect on ion transport, increased cAMP to the same level as did CTm, and the effect on cAMP of adding toxin to both sides was additive. We conclude that 1) active secretion is probably stimulated by cholera toxin added on the serosal side, although more slowly than after addition to the mucosal side and 2) much of the toxin-stimulated cAMP content of the mucosa is not coupled to secretion.

Catecholamine effects on cyclic AMP levels and ion secretion in rabbit ileal mucosa.

Effects of catecholamines on cyclic AMP (cAMP) levels and ion fluxes were examined in isolated rabbit ileal mucosa. The base-line cAMP level was unaffected by epinephrine (Epi), norepinephrine (Norepi), and isoproterenol. The theophylline-augmented cAMP level was decreased slightly be Epi in one series of experiments but not in another. Propranolol did not enhance this effect. The increase in cAMP level produced by cholera toxin was almost completely reversed by addition of Epi or Norepi. This reversal was prevented by phenoxybenzamine. Epi also partially reversed the increase in cAMP level produced by prostaglandin E1. Effects of Epi on ion fluxes were determined following addition of secretagogues. Epi significantly decreased theophylline-induced but not cAMP or cholera toxin-induced Cl secretion. A decrease in short-circuit current was nonetheless observed in the latter two instances. The observed discrepancies between alpha-adrenergic effects on cAMP levels and ion fluxes suggest the following possibilities: 1) ion transport-related cAMP is only a small fraction of total mucosal cAMP; 2) cAMP-induced active ion secretion is only slowly reversible, or 3) effects of alpha-adrenergic stimuli on ion transport are not due to inhibition of cAMP accumulation.

Ileal HCO3 secretion: relationship to Na and Cl transport and effect of theophylline.

Interrelationships among Na, Cl, and HCO3 transport processes were examined in short-circuited rabbit ileal mucosa. As serosal (HCO3) was increased from 10 to 50 mM (pH from 7.1 to 7.8), net Na absorption decreased from 4.6 to 0.3 mueq/h-cm2, net Cl flux changed from absorption of 0.9 to secretion of 0.9 and a net HCO3 secretion of 3.0 developed. A similar change in net Cl flux was also observed when serosal Pco2 was altered at constant (HCO3). In Cl-free SO4-Ringer, serosal alkalinization produced net HCO3 secretion which was not significantly less than that observed in Cl-containing Ringer. Theophylline caused secretory changes in net Na and Cl fluxes at both 10 and 50 mM serosal (HCO3). Theophylline did not alter net HCO3 flux in Cl-Ringer but increased net HCO3 flux in SO4-Ringer. Total dc conductance was decreased by both serosal alkalinization and theophylline. Shortcircuit current was consistently increased by theophylline but not by serosal alkalinization. The results indicate that ileal ion transport is regulated in part by serosal pH and/or (HCO3) and that resulting changes in Cl and HCO3 transport are coupled one-for-one with changes in Na transport. Furthermore, HCO3 secretion does not require the presence of Cl in the bathing medium.

Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa.

Vasoactive intestinal peptide (VIP), originally isolated from hog small intestinal mucosa, has been shown to cause small intestinal secretion. More recently, this peptide has been identified in the plasma and tumors of patients with the so-called "pancreatic cholera" syndrome. In order to explore the possible role of VIP in the pathogenesis of this syndrome, we examined the effects of this peptide and other hormones on the cyclic AMP levels, adenylate cyclase activity, and ion transport in in vitro preparations of ileal mucosa. In rabbit ileal mucosa, VIP (20 mug/ml) caused a prompt fivefold increase in cyclic AMP level, whereas nine other hormones, which have been postulated to cause intestinal secretion, failed to exert such an effect. Pentagastrin and glucagon also failed to increase cyclic AMP levels in canine ileal mucosa. An increase in mucosal cyclic AMP levels was observed at a VIP concentration of 0.1 mug/ml and appeared to be nearly maximal at 2.0 mug/ml. VIP (100 mug/ml) stimulated adenylate cyclase activity in a membrane preparation from rabbit ileal mucosa. Secretin (6.0 x 10(-5) M) failed to do so. When added to the serosal side of isolated rabbit ileal mucosa clamped in an Ussing chamber, VIP (2 mug/ml) increased short-circuit current (SCC) and caused net secretion of both Cl and Na. Net Cl secretion exceeded net Na secretion. These effects of VIP on mucosal cyclic AMP metabolism and ion transport are similar to those observed with cholera enterotoxin and certain prostaglandins. VIP was also tested with normal human ileal mucosa. At a concentration of 2 mug/ml it caused a fivefold increase in cyclic AMP level and an increase in SCC of the same magnitude as that caused by 5 mM theophylline. Addition of a second 2-mug/ml dose of VIP and addition of theophylline after VIP produced no further change in SCC. We conclude the VIP stimulates adenylate cyclase and active ion secretion in both rabbit and human ileal mucosa. This may be related to the pathogenesis of diarrhea in patients with the pancreatic cholera syndrome.