Methylation increases sodium transport into A6 apical membrane vesicles: possible mode of aldosterone action.

When isolated apical membrane vesicles prepared from cultured A6 epithelia were incubated in vitro with the methyl donor S-adenosylmethionine, the control rate of amiloride-inhibitable sodium transport was doubled. The methylation inhibitors 3-deazaadenosine and S-adenosyl homocysteine returned the S-adenosyl-methionine-stimulated sodium transport to control levels. Neither these agents nor adenosine affected sodium transport into control vesicles. In vesicles incubated with S-adenosyl-[3H-methyl]methionine, both membrane phospholipids and proteins were labeled, and this labeling was inhibited by deazaadenosine. In vesicles prepared from A6 cells treated with aldosterone, sodium transport was twice the control value and S-adenosylmethionine did not cause any further stimulation of transport. In those vesicles, both lipid and protein methylation were increased. These results suggest that methylation, which increases the rate of amiloride-sensitive sodium transport is involved in the action of aldosterone at the apical membrane level in epithelia.

Cholinergic agonists stimulate calcium uptake and cGMP formation in human erythrocytes.

Human erythrocytes possess a muscarinic cholinergic receptor sensitive to cholinergic agonists which stimulate transient increases in calcium uptake and subsequent cyclic GMP formation. These phenomena can be blocked by atropine and EGTA. The cholinergic stimulation of cyclic GMP formation depends on Ca2+ uptake from external media. The effects of cholinergic agonists on the erythrocyte resemble their effect on calcium channels in nervous tissue. The cholinergic stimulation of Ca2+ uptake in erythrocytes may affect the calcium-sensitive mechanism involved in the shape, permeability and rigidity of these cells.

The effect of hypothermia on potassium and glucose changes in isobaric hemorrhagic shock in the rat.

Hypothermia has been shown to decrease oxygen consumption requirements and improve survival during hemorrhagic shock. however, hypothermia applied therapeutically does not prevent the development of a lactic acidosis during hemorrhage. We re-examined the development of a hemorrhage-induced lactic acidosis and other metabolic parameters (glucose, plasma electrolytes, and arterial blood gases) at various temperatures (29-37 degrees C) to better define the protective action of hypothermia in hemorrhagic shock. Five groups of male, Sprague-Dawley rats were bled to a mean arterial blood pressure (MABP) of 40 mmHg over a 15 min period and held there by further blood removal until death. The final level and rate of development of the lactic acidemia was the same in all groups. However, the rate of decline in plasma glucose and rate of rise in plasma potassium were temperature dependent. These results suggest that temperature-dependent changes in serum glucose and potassium may contribute to the protective effect of hypothermia during hemorrhagic shock.

The role of histamine in mediating the decompensatory phase of hemorrhagic shock in the rat.

Our laboratory has previously reported that plasma histamine levels rise significantly and coincidentally with the onset of the decompensatory phase of isobaric hemorrhagic shock in rats. The histamine levels seen in shock were comparable to those that induce profound vasodilatation in many vascular beds under normovolemic conditions. We, therefore, sought to determine whether the elevation in plasma histamine contributes to the cardiovascular collapse seen in the decompensatory phase of hemorrhagic shock. Sprague-Dawley rats were bled according to an isobaric bleeding protocol which maintained the mean arterial blood pressure (MAP) at 40 mmHg until death. Selected H1 (diphenhydramine) and/or H2 (cimetidine and famotidine) antagonists were administered at 75% of the estimated peak shed blood volume (PSBV), a point preceding the rise in plasma histamine. Plasma histamine levels in all groups were similar throughout the time course of hemorrhagic shock. None of the histamine receptor antagonists affected the time of onset or the rate of decompensation. Suspecting that hypotension may alter the animal's response to histamine, we investigated the effect of exogenous histamine administration on MAP before and after hemorrhage. In unbled animals, bolus histamine infusions (.6 mg/kg) dropped the MAP by 62.0 +/- 2.7 mmHg, however, in animals bled to 40 mmHg, histamine dropped the MAP by 7.2 +/- 2.7 mmHg (p = .002). On the basis of the results of these two interventions, we conclude that histamine is not an important mediator of the cardiovascular collapse seen in the decompensatory phase of hemorrhagic shock in the rat.

Plasma and tissue histamine changes during hemorrhagic shock in the rat.

We investigated the phase-associated changes in plasma histamine levels in an isobaric model of hemorrhagic shock, in an attempt to determine whether histamine might be an etiologic factor in the onset of decompensation. Sprague-Dawley rats were bled according to an isobaric bleeding protocol which maintained the mean arterial blood pressure at 40 mmHg until death. The status of vascular compensation for the blood loss was tracked by measurement of the shed blood volume (SBV) required to maintain the target pressure. Blood samples for analysis were taken at the control period and at 25% intervals of the peak shed blood volume (PSBV) during the compensatory and decompensatory phases. Plasma and tissue histamine levels were measured using a radioimmunoassay method. In untreated animals, plasma histamine levels at control, 75 and 100% of the PSBV, and after return of 25 and 75% of the PSBV were 45 +/- 10, 48 +/- 9,134 +/- 48,693 +/- 351, and 994 +/- 371 nM, respectively. These results show that rises in plasma histamine occurred coincidentally with the onset of decompensation (p < .05), however, the subsequent rate of decompensation did not correlate with plasma histamine changes during decompensation. Organ histamine levels measured after hemorrhage were lower in the duodenum and colon than in unbled control animals, suggesting that parts of the intestinal tract may contribute to the elevated plasma histamine levels seen in severe hypotension (p < .05).

Effect of sodium ions on calcium movements in isolated synaptic terminals.

Calcium accumulation has been studied in isolated presynaptic nerve terminals from rat brain. Calcium influx is increased when external sodium is replaced by lithium or choline, or when the internal sodium concentration is increased by treatment with ouabain. Calcium efflux is reduced when external sodium is replaced by lithium. The lithium-stimulated calcium accumulation appears to be associated with the nerve terminals, and not with mitochondria or membrane fragments. It is dependent upon an osmotically sensitive surface membrane. The results are consistent with the hypothesis that calcium is actively extruded from mammalian central neurons by a mechanism which exchanges calcium for sodium, and which derives its energy from the sodium concentration gradient.

Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells.

Aldosterone and insulin stimulate Na+ transport through mechanisms involving protein synthesis. Na+-K+-ATPase has been implicated in the action of both hormones. We examined the effect of aldosterone and insulin on Na+-K+-ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (ISC) in TB6C cells. Aldosterone increases Na+-K+-ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in ISC, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase ISC in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na+-K+-ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na+ entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na+-K+-ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on ISC.

Effect of aldosterone on potassium transport in the toad bladder.

The effect of aldosterone on potassium uptake by the toad bladder is described. The hormone stimulated the uptake of potassium across the serosal border of the bladder. The increased uptake was the consequence of an increase in the rate of potassium influx. An effect on potassium uptake was characterized by a latent period of approximately 60 min; it was evident for periods as long as 5 h, and it was abolished by addition of actinomycin D. The time course of the aldosterone effect on potassium closely resembled the effect of the hormone on sodium transport. It is suggested that aldosterone influences potassium transport in the toad bladder via DNA-dependent RNA synthesis. In addition, it is suggested that the effect of the hormone on potassium and sodium may be in some way related.

Effects of insulin, ADH, and cyclic AMP on sodium transport in the toad bladder.

These studies further define the mechanisms by which insulin stimulates Na transport in the toad bladder. Serosal but not mucosal addition of insulin, 100-1,000 mU/ml, stimulated short-circuit current (SCC) by 25-50%. The initial rise in SCC occurred at 5 min and the peak response at 15-25 min. Doses of insulin greater than 250 mU/ml increased SCC values for up to 3 h. Actinomycin D did not block the early rise in SCC produced by insulin, but it blocked the delayed effects. Insulin increased SCC in substrate-depleted bladders, although the increase in SCC was less (P less than 0.01) than in nonsubstrate-depleted bladders. Pyruvate addition to substrate-depleted bladders restored to normal the rise in SCC observed after insulin. Simultaneous addition of ADH and insulin led to an increase in SCC that was greater than the sum of the responses observed when each hormone was added independently. Synergistic effects on SCC were also obtained with cyclic AMP and insulin. Insulin did not increase cyclic AMP levels in toad bladder epithelial cells. It is suggested that insulin stimulates active Na transport by two mechanisms: 1) a rapid phase, which may involve unmasking of pump sites within the membrane, and 2) a delayed effect which seems to require protein synthesis. The synergism of which seems to require protein synthesis. The synergism of insulin with ADH or cyclic AMP may reflect a facilitative effect of insulin on ADH or cyclic AMP-sensitive pump sites or, alternatively, the uncovering of latent pump sites that then may be available to stimulation by ADH or cyclic AMP.

Reversible defect in cAMP metabolism in lymphocytes in malaria infection.

Peripheral blood lymphocytes were observed to have a defect in adenosine 3',5'-monophosphate (cAMP) metabolism during acute malaria infection which reversed once parasites were eliminated from the host circulation. The defect was characterized by decreased intracellular cAMP levels in lymphocytes and by hyporesponsiveness to adenosine or forskolin stimulation of cAMP production. These biochemical changes appeared to correlate functionally with a reduction in the proliferative response of lymphocytes to concanavalin A. A defect in the second messenger role of cAMP in immune effector cells may underlie immunosuppression in malaria infection.

Aldosterone-stimulated transmethylations are linked to sodium transport.

The effect of aldosterone (Aldo) on phospholipid (PL) biosynthesis in cultured toad bladder epithelial cells was studied in cells incubated with [1,2-14C]choline and [methyl-3H]methionine over a 5-h period. Aldo (10(-7) M) did not alter the uptake of either precursor but significantly stimulated the incorporation of both labels into phosphatidylcholine (PC), the only PL labeled. 3H labeling of PC increased 29% and 14C incorporation into PC increased 34% in cells exposed to Aldo. A similar 30% increase in protein carboxymethylation occurred in cells treated with Aldo. 3-Deazaadenosine (DZA), a methylation inhibitor, abolished the Aldo-stimulated increase in PC labeling from [3H]methionine. PC labeling from [1,2-14C]choline was not affected by DZA. Basal and Aldo-stimulated protein carboxy-methylation were inhibited by DZA. DZA (300 microM) caused a mild decrease in basal short-circuit current (ISC) but completely inhibited the ISC response to 10(-7) M Aldo. Inhibition was complete when DZA was added up to 2 h following exposure to Aldo, and was reversible. Cells previously exposed to Aldo showed a significant increase in ISC within 2 h following removal of DZA. We conclude that Aldo stimulates PL methylation, protein carboxymethylation, and turnover of PC from choline. Inhibition of methylation reactions coincides with the inhibition of ISC response to Aldo.

Human malaria parasite adenosine deaminase. Characterization in host enzyme-deficient erythrocyte culture.

Human malaria infected erythrocytes show a dramatic increase in adenosine deaminase activity in vitro. Using recently developed culture techniques, adenosine deaminase-deficient human erythrocytes were infected in vitro with the major human pathogen Plasmodium falciparum. Adenosine deaminase activity was undetectable in the uninfected host red cells, but increased by 2-fold over normal levels in these cells with an 8% parasitemia. The enzyme in these cells appeared unique in that its activity was markedly elevated over that of other parasite purine enzymes, was not cross-reactive with antibody against human erythrocyte adenosine deaminase, and though inhibited competitively by deoxycoformycin was relatively insensitive to erythro-9-(2-hydroxy-3-nonyl) adenine. The use of adenosine deaminase-deficient erythrocytes for the in vitro cultivation of Plasmodium provides a unique system for the study of parasite enzyme and allows further insight into the purine metabolism of the intraerythrocytic malaria parasite.

Hypertonic saline resuscitation detrimentally affects renal function and survival in dehydrated rats.

The purpose of our studies was to determine whether hypertonic (7.5%) saline (HTS) resuscitation is effective in the setting of dehydration. We compared the effects of HTS (5 cc/kg) to those of Ringer's lactate (RL; 45 cc/kg) on renal function, following resuscitation from hypovolemia in hydrated (free access to food/water) vs. dehydrated (food/water restricted) rats (300-350 g). Renal failure was produced by hemorrhage (15 cc/kg) plus renal artery occlusion (25 min) followed by fluid resuscitation. Dehydration was confirmed by hemoconcentration and weight loss (8-10%). Renal function was assessed at 24 hr using 14C-inulin clearance (Cin) measurements. In hydrated animals, the Cin of RL-treated rats (625 +/- 54 microliters/min/100 g; n = 12) was no different from the Cin in HTS-treated rats (517 +/- 48 microliters/min/100 g; n = 13). Among dehydrated rats, Cin in HTS-treated rats (n = 6) was significantly lower (P < or = 0.05) than in RL-treated rats (n = 5) (117 +/- 33 microliters/min/100 g vs. 542 +/- 84 microliters/min/100 g, respectively). Cin in dehydrated RL-treated rats was not significantly different from that in hydrated RL-treated rats. Furthermore, in dehydrated animals, nine of nine resuscitated with RL survived, compared to six of 13 resuscitated with HTS. All hydrated animals survived. In summary, renal failure was ameliorated by RL and worsened by HTS resuscitation in dehydrated rats. Furthermore, mortality was increased in dehydrated animals resuscitated with HTS compared to RL.

Paradoxical effects of adenosine on neutrophil chemotaxis.

Chemotaxis of rabbit neutrophils is most sensitive to inhibition by 3-deazaadenosine, followed by 3-deaza-(+/-)aristeromycin, 3-deaza-(+/-)aristeromycinylhomocysteine, 3-deazaadenosylhomocysteine, and adenosylhomocysteine, in that order. Although adenosine by itself had no effect on the chemotaxis of neutrophils, it essentially abolished the inhibitory effects of 3-deaza-adenosine on chemotaxis and the reduction of nitroblue tetrazolium. Paradoxically, adenosine enhanced the inhibition of chemotaxis by 3-deazaadenosylhomocysteine slightly and that of 3-deaza-(+/-)aristeromycin significantly. Adenosine alone unexpectedly inhibited phospholipid methylation to the same extent as 3-deazaadenosine, and reduced protein carboxymethylation to a lesser degree. The inhibition of these two methylation reactions by 3-deazaadenosine was, however, not substantially altered in the presence of adenosine. Drastic changes in the ratio of adenosylmethionine/nucleosidylhomocysteine were observed in the presence of adenosine, 3-deazaadenosine, 3-deaza-(+/-)aristeromycin, or of adenosine in combination with each of the latter compounds. There was no significant effect on the binding of chemotactic peptide to receptors, or on the ratio of ATP/ADP in cells treated by the analogs. These results suggest that the inhibition of methylation reactions per se is not enough to account for the inhibition of both chemotaxis and the reduction of nitroblue tetrazolium by neutrophils.

Prolonged alteration in gut permeability following nonthermal injury.

To assess the extent of intestinal permeability following nonthermal injury ratios of lactulose and mannitol (L-M) concentrations in urine following enteral administration were determined simultaneously by a gas-liquid chromatography assay on days 3, 5, 7, 10, and 13 in 15 patients with an Injury Severity Score > 20. Thirteen of 15 patients recovered uneventfully and two developed minor infections. L-M ratios were significantly increased on days 3-10 (P < 0.05 vs controls). The data are consistent with previous studies describing early changes in gut permeability following nonthermal injury and show that altered permeability can persist for up to 10 days in patients with uneventful recoveries.

Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors.

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.