Relationship between membrane potential changes and superoxide-releasing capacity in resident and activated mouse peritoneal macrophages

In an attempt to understand better the molecular basis for the enhanced respiratory burst of activated macrophages (M phi), we investigated the relationship between stimulus-induced changes in membrane potential and release of superoxide anion (O2-) in mouse peritoneal M phi. Resident M phi and M phi elicited by injection of lipopolysaccharide (LPS-M phi) or obtained from animals infected with bacille Calmette-Guérin (BCG-M phi) were used. LPS-M phi and BCG-M phi showed more pronounced changes in membrane potential (depolarization) and greater release of O2- on contact with phorbol myristate acetate (PMA) than did resident macrophages. The lag time between addition of stimulus and onset of release of O2- was reduced in activated compared with resident cells. Membrane potential changes began 60 to 90 sec before release of O2- could be detected in each cell type. The dose-response curves for triggering of membrane potential changes and O2- release by PMA were identical. The magnitude of membrane potential changes and of O2- release in LPS-M phi and BCG-M phi declined progressively during in vitro culture, and values on day 3 approached those in resident macrophages ("deactivation"). Extracellular glucose was required for effective stimulated change in membrane potential and O2- release. These findings indicate that membrane potential changes are closely associated with O2- -releasing capacity in macrophages, and that the systems that mediate membrane potential changes and production of O2- develop or decline concomitantly during activation or deactivation of the cells. Although the plasma membrane was highly depolarized by high extracellular K+ or by the sodium ionophore gramicidin, O2- release was not induced by these maneuvers, indicating that changes in membrane potential by themselves are not sufficient to trigger the respiratory burst in macrophages. Release of O2- was not impaired in buffers in which Na+ was completely replaced with equimolar concentrations of K+ or choline+; thus, induction or maintenance of the respiratory burst in M phi does not require an influx of Na+.     

Failure of Trypanosoma cruzi to trigger the respiratory burst of activated macrophages. Mechanism for immune evasion and importance of oxygen-independent killing

Activated macrophages are thought to kill Trypanosoma cruzi, which lack catalase, by the generation of hydrogen peroxide. We investigated triggering of the respiratory burst of activated macrophages induced by phagocytosis of virulent T. cruzi, bloodform trypomastigotes, amastigotes obtained from spleens, and tissue culture organisms; and of relatively nonvirulent epimastigotes. All stages of T. cruzi prompted the release of less than 10% of hydrogen peroxide released by activated macrophages when stimulated with PMA or Candida. Superoxide anion production was not stimulated by PMA or Candida in activated macrophages nor was there a significant qualitative reduction of nitroblue tetrazolium induced by ingestion of virulent T. cruzi. Opsonization of T. cruzi with specific antibody did not promote the release of hydrogen peroxide or the reduction of nitroblue tetrazolium. Similar results were observed with activated spleen macrophages. Incubation of activated macrophages with catalase, catalase and superoxide dismutase, sodium benzoate with or without catalase, and respiratory burst-exhausting PMA failed to inhibit the killing of T. cruzi in vitro. These results indicate that 1) virulent opsonized or unopsonized T. cruzi do not trigger a respiratory burst by activated macrophages and 2) oxygen-independent killing of T. cruzi is of prime importance.     

A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils

A Na+/Ca2+ exchange mechanism has been recently described in human neutrophils that constitutes the principal pathway for Ca2+ influx into resting cells. The potential role of this system in regulating the respiratory burst in response to activation by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine was explored. In the presence of 1 mM Ca2+, a variety of di- and trivalent cations suppressed the generation of O(-2) radicals in a series of decreasing efficacy: La3+ approximately Zn2+ much greater than Sr2+ approximately Cd2+ greater than Ba2+ greater than Co2+ greater than Ni2+ approximately Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na+/Ca2+ counter-transport. Benzamil, phenamil, and 2',4'-dichlorobenzamil, analogues of amiloride which selectively block Na+/Ca2+ exchange in neutrophils, likewise suppressed the release of O(-2) with apparent Ki values of approximately 30 microM. The effect of the cations was competitive with Ca2+, while the interaction between the benzamil derivatives and Ca2+ appeared to be noncompetitive in nature. Both the divalent cations and benzamil also inhibited the rise in cytoplasmic Ca2+ as monitored by fura-2 fluorescence: these agents reduced peak cytosolic Ca2+ levels after N-formyl-methionyl-leucyl-phenylalanine stimulation to values seen in the absence of extracellular Ca2+. These results are compatible with the hypothesis that the influx of Ca2+ via Na+/Ca2+ exchange contributes to the transient elevation in intracellular free Ca2+. The polyvalent cations block the entry of critical Ca2+ ions by competing with Ca2+ for binding to the translocation site on the exchange carrier, while benzamil acts by lowering the maximal transport rate. These studies emphasize that Na+/Ca2+ exchange through its effects on cytoplasmic Ca2+ plays a major regulatory role in activation of the respiratory burst in chemotactic factor-stimulated neutrophils.     

Mycobacterium lepraemurium activates macrophages but fails to trigger release of superoxide anion

Mycobacterium lepraemurium failed to stimulate a normal respiratory burst when presented to mouse peritoneal or bone marrow macrophages. By comparison, Mycobacterium bovis (strain Bacillus Calmette-Guerin) or Saccharomyces cerevisiae, as expected, stimulated macrophages to release a large amount of superoxide anion (O2-). M. lepraemurium did not interfere with the response to yeast when both microbes were added together to macrophages. The low release of O2- induced by M. lepraemurium was not due to failure of M. lepraemurium to activate or prime macrophages, because exposure of macrophages to M. lepraemurium caused the expected enhancement of O2- release when the macrophages were stimulated by PMA. Similarly, macrophages taken from mice infected with M. lepraemurium were activated, as indicated by high PMA-stimulated O2- release. Macrophages primed in vitro by exposure to Escherichia coli LPS for 24 h did show a moderate O2- response when stimulated by M. lepraemurium, but macrophages primed by exposure to IFN-gamma muramyl dipeptide, or M. lepraemurium showed a weak response when subsequently challenged with M. lepraemurium. The priming effect of M. lepraemurium or LPS decreased substantially after macrophages were cultured in fresh medium for 24 h. Heat killing or opsonization of M. lepraemurium caused the M. lepraemurium to stimulate a high amount of O2- release from LPS-primed macrophages, but heat killing or opsonization of M. lepraemurium had no effect on release of O2- from unprimed macrophages. The results suggest that M. lepraemurium is taken into macrophages by a mechanism that bypasses the FcR and other receptors that are capable of triggering the production of O2-.     

Hyperoxia alters effect of calcium on rat alveolar macrophage superoxide production

The effect of hyperoxia on the Ca2+ dependence of stimulated superoxide anion radical (O2-.) production (the respiratory burst) of rat alveolar macrophages was investigated. Enhancement of the concanavalin A (con A)-stimulated respiratory burst by extracellular Ca2+ was suppressed by O2 exposure. Similarly, the inhibitory effect of verapamil on the con A-stimulated respiratory burst was reduced by O2 exposure. O2 exposure also inhibited con A stimulation that was independent of Ca2+ entry. Exposure to O2 also caused a decline in O2-. production stimulated by either A23187 or phorbol myristate acetate (PMA). With A23187 stimulation, extracellular Ca2+ was essential for either air-exposed (control) or O2-exposed cells. With PMA, stimulation was independent of extracellular Ca2+ for either air or O2-exposed macrophages and verapamil did not inhibit. Free intracellular Ca2+ concentration ([Ca2+]i) was measured in control and O2-exposed alveolar macrophages. Hyperoxic exposure did not alter [Ca2+]i in unstimulated cells. In controls, con A stimulated an immediate increase in [Ca2+]i followed by a rapid decrease and a second rise and fall. The second elevation was suppressed by verapamil or ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid or O2 exposure. The results of both the respiratory burst assays and measurement of con A-stimulated changes in [Ca2+]i suggest that Ca2+ entry involved in stimulus-response coupling is suppressed in cellular O2 toxicity.     

Na+,K+ pump and Na+-coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C.

This review updates our current knowledge on the regulation of Na+/H+ exchanger, Na+,K+,Cl- cotransporter, Na+,Pi cotransporter, and Na+,K+ pump in isolated epithelial cells from mammalian kidney by protein kinase C (PKC). In cells derived from different tubule segments, an activator of PKC, 4beta-phorbol 12-myristate 13-acetate (PMA), inhibits apical Na+/H+ exchanger (NHE3), Na+,Pi cotransport, and basolateral Na+,K+ cotransport (NKCCl) and augments Na+,K+ pump. In PMA-treated proximal tubules, activation of Na+,K+ pump probably plays a major role in increased reabsorption of salt and osmotically obliged water. In Madin-Darby canine kidney (MDCK) cells, which are highly abundant with intercalated cells from the collecting duct, PMA completely blocks Na+,K+,Cl- cotransport and decreases the activity of Na+,Pi cotransport by 30-40%. In these cells, agonists of P2 purinoceptors inhibit Na+,K+,Cl- and Na+,Pi cotransport by 50-70% via a PKC-independent pathway. In contrast with MDCK cells, in epithelial cells derived from proximal and distal tubules of the rabbit kidney, Na+,K+,Cl- cotransport is inhibited by PMA but is insensitive to P2 receptor activation. In proximal tubules, PKC-induced inhibition of NHE3 and Na+,Pi cotransporter can be triggered by parathyroid hormone. Both PKC and cAMP signaling contribute to dopaminergic inhibition of NHE3 and Na+,K+ pump. The receptors triggering PKC-mediated activation of Na+,K+ pump remain unknown. Recent data suggest that the PKC signaling system is involved in abnormalities of dopaminergic regulation of renal ion transport in hypertension and in the development of diabetic complications. The physiological and pathophysiological implications of PKC-independent regulation of renal ion transporters by P2 purinoceptors has not yet been examined.     

Phorbol ester-stimulated superoxide production by murine bone marrow-derived macrophages requires preexposure to cytokines

Murine resident peritoneal macrophages (RPM) generate superoxide (O2-) in response to stimulation with PMA or zymosan. Murine bone marrow-derived macrophages (BMM) generate O2- in response to zymosan but not PMA. However, the ability to generate O2- in response to PMA could be induced in BMM by pre-exposing the cells to certain cytokines, including granulocyte-macrophage CSF (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, and, to a lesser extent, IL-1 alpha. Bacterial LPS also induced the ability to respond to PMA. These same agents were also shown to prime RPM for enhanced PMA-induced respiratory burst. In contrast to GM-CSF, CSF-1 did not enhance the ability of BMM or RPM to generate O2- in response to PMA. Pretreatment with GM-CSF or TNF-alpha did not significantly affect the zymosan-induced release of O2- by BMM. These results suggest that unprimed BMM have a deficiency in the PMA-dependent signaling pathway that is corrected by exposure to selected cytokines. The results also raise the possibility that the basal ability of tissue macrophages to generate a respiratory burst in response to PMA may be a reflection of in vivo exposure to cytokines.     

Macrophage colony-stimulating factor (M-CSF) enhances the capacity of murine macrophages to secrete oxygen reduction products

The capacity of macrophage colony-stimulating factor (M-CSF) to enhance respiratory burst activity in peritoneal macrophages was measured. Macrophages incubated for 48 hr or more with concentrated L cell-conditioned medium as a source of M-CSF released two to three times as much O2- in response to PMA as did unexposed macrophages. Stimulation was noted at concentrations of colony-stimulating activity from 0.1 to 2000 U/ml and was maximal at 10 to 100 U/ml. Purified, endotoxin-free CSF enhanced secretion to a similar degree as unpurified L cell-conditioned medium. Release of O2- by M-CSF macrophages occurred over 60 min and was triggered by opsonized zymosan as well as PMA. H2O2 release was also enhanced in macrophages exposed to both unpurified and purified M-CSF. These data indicate that M-CSF enhances the capacity of mature macrophages to release oxygen reduction products, and they are consistent with reports that CSF can stimulate the release of other secretory products.     

Impairment of Na+/H+ exchange underlies inhibitory effects of Na+-free media on leukocyte function

Inhibition of activation has been reported when neutrophils are suspended in Na+-free media. We considered the possibility that impairment of cellular pH (pHi) regulation due to elimination of Na+/H+ exchange underlies this effect. In the absence of Na+, the phorbol ester-induced respiratory burst was partially inhibited and a concomitant cytoplasmic acidification recorded. Using nigericin/K+ to clamp pHi we demonstrated that the acidification accounts for the inhibition of O2 uptake. Moreover, in Na+-free media, relieving the acidification by means of ionophores restored maximal O2 consumption. It was concluded that Na+ is not directly involved in signal transduction during stimulation. Instead, omission of Na+ affects neutrophils activation indirectly, by impairing pHi regulation.     

Synergistic Effect of Prostaglandin E2 and Ouabain on Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells

We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+,K+-ATPase, synergistically induced a gradual secretion of catecholamines from the cells. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1 = E2 greater than F2 alpha greater than D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+-depleted medium and not observed in Ca2+-free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose-dependent manner with half-maximal stimulation at 1 microM, and omission of K+ from the medium, a condition which suppresses the Na+,K+-ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose-dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+-antiport, but not by the Na+-channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 microM inhibited PGE2-enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+-antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+-free medium. These results taken together indicate that PGE2 activates the Na+,H+-antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+,K+-ATPase and activation of Na+,H+-antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

Properties of a Sodium Channel (Nax) Activated by Strong Depolarization of Xenopus Oocytes

Short (<1 sec) duration depolarization of Xenopus laevis oocytes to voltages greater than +40 mV activates a sodium-selective channel (Na(x)) with sodium permeability five to six times greater than the permeability of other monovalent cations examined, including K+, Rb+, Cs+, TMA+, and Choline+. The permeability to Li+ is about equal to that of Na+. This channel was present in all oocytes examined. The kinetics, voltage dependence and pharmacology of Na(x)distinguish it from TTX-sensitive or epithelial sodium channels. It is also different from the sodium channel of Xenopus oocytes activated by prolonged depolarization, which is more highly selective for Na+, requires prolonged depolarization to be activated, and is blocked by Li+. Intracellular Mg2+ reversibly inhibits Na(x), whereas extracellular Mg2+ does not have an inhibitory effect. Intracellular Mg2+ inhibition of Na(x), is voltage dependent, suggesting that Mg2+ binding occurs within the membrane field. Eosin is also a reversible voltage-dependent intracellular inhibitor of Na(x), suggesting that a P-type ATPase may mediate the current. An additional cytoplasmic factor is involved in maintaining Na(x) since the current runs down in internally perfused oocytes and excised membrane patches. The rundown is reversible by reintroduction of the membrane patch into oocyte cytoplasm. The cytoplasmic factor is not ATP, because ATP has no effect on Na(x) current magnitude in either cut-open or inside-out patch preparations. Extracellular Gd3+ is also an inhibitor of Na(x). Na(x) activation follows a sigmoid time course. Its half-maximal activation potential is +100 mV and the effective valence estimated from the steepness of conductance activation is 1.0. Na(x) deactivates monoexponentially upon return to the holding potential (-40 mV). The deactivation rate is voltage dependent, increasing at more negative membrane potentials.     

Rat macrophage treatment with lipopolysaccharide leads to a reduction in respiratory burst product secretion and a decrease in NADPH oxidase affinity

The effect of LPS on the respiratory burst in resident rat peritoneal macrophages has been examined. Rat macrophages secreted high levels of both O2- and H2O2 in response to triggering with phorbol esters, opsonized zymosan, and immune complexes. After culture in vitro with LPS these macrophages exhibited a marked diminution in their capacity to secrete high levels of respiratory burst products. The LPS-mediated loss of secretory activity was apparent after 2 hr of exposure to LPS and was inhibitable by polymyxin B in a dose-dependent fashion. The effect was not selective for any triggering agent type as inhibition of secretory activity occurred after triggering with PMA, zymosan and immune complexes. PGE2 added at levels secreted by the macrophages in response to LPS also inhibited respiratory burst product secretion. In addition, indomethacin prevented the LPS-mediated inhibition of secretion. Because the inhibition of secretion was common to all triggering agents tested, this suggested that the basis for the impaired secretion was at a level other than the receptor for the triggering agent. Both LPS and PGE2 treatment of the macrophages increased the Km of the oxidase for NADPH (1.7- to 2.3-fold) without affecting significantly the Vmax of the enzyme. These data suggest that stimulation of rat peritoneal macrophages by LPS results in an impaired ability to secrete respiratory burst products as a result of a PGE2-mediated decrease in NADPH oxidase affinity and that this alteration is independent of alterations in tumoricidal activity.     

Formyl peptide stimulation of superoxide anion release from lung macrophages: sodium and potassium involvement

We examined the role of the monovalent cations Na+ and K+ in the events encompassing the release of O-2 by alveolar macrophages after stimulation with formyl methionyl phenylalanine (FMP). This was accomplished by determining the effect of changing the extracellular [Na+] and/or [K+] on FMP-stimulated O-2 production; and measuring 22Na+, 42K+ and 86Rb+ influx and efflux and intracellular [K+] for control and FMP-stimulated alveolar macrophages. Stimulated O-2 production was relatively insensitive to changes in extracellular K+ or Na+ concentrations until the [Na+] was decreased below 35 mM. At 4 mM [Na+], the rate of O-2 production remained at 75% of the maximal rate observed at physiological concentrations of [Na+]. Both influx and efflux of 22Na+ were stimulated above control rates by FMP. The increased rates of fluxes lasted for a few minutes suggesting a transient increase in membrane permeability to Na+. Ouabain partially inhibited 22Na+ efflux but had no effect on O-2 release. The influx of 86Rb+ and 42K+ was not altered by the addition of FMP but was virtually abolished in the presence of 10 microM ouabain or 1 mM quinine. In the presence of extracellular calcium, FMP-stimulated a prolonged (greater than 20 minutes) increase in 86Rb+ or 42K+ efflux which was inhibitable by 1 mM quinine. In the absence of extracellular calcium, FMP stimulation of K+ efflux was greatly diminished and was not affected by quinine, although quinine still inhibited O-2 production under these conditions. It was also observed that there was a loss of intracellular K+ when cells were stimulated by FMP in the presence of Ca+2, but not in the absence of Ca+2. Taken together, these results suggest a minimal direct role, if any, for K+ in the events that lead to FMP-stimulated O-2 release by alveolar macrophages.     

Effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on transmembrane electrical potentials in granulocytes: relationship between enhancement of ligand-mediated depolarization and augmentation of superoxide anion (O2-) production

When human granulocytes that have been primed with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) are activated by ligands that stimulate the respiratory burst, the amount of superoxide anion (O2-) they generate is significantly increased. We have found that the accelerated rate of O2- release occurring under these conditions is accompanied by an antecedent increase in membrane depolarization. We examined the nature of the enhancement of membrane depolarization in GM-CSFrh-primed granulocytes and investigated its relationship to the increase in O2- generation by N-formyl methionylleucylphenylalanine (fMLP)-activated granulocytes. We found that augmented depolarization could not be accounted for by a change in the resting membrane potential induced by the growth factor and was still present after either blocking passive transmembrane Na+ movement with dimethylamiloride or by increasing the membrane's permeability to K+ with valinomycin. When their ability to depolarize was virtually eliminated by dissipating the transmembrane K+ gradient, GM-CSFrh-pretreated cells continued to generate more O2- after fMLP than did control cells. These results indicate that augmentation of the granulocyte's ability to generate O2- anions, which is induced by priming with GM-CSFrh, is independent both of the resting transmembrane potential and of alterations in the extent of membrane potential change induced by stimuli such as fMLP.     

Binding of somatostatin to guinea-pig pancreatic membranes: regulation by ions

Somatostatin receptors were characterized on guinea-pig pancreatic acini membranes using 125I-[Tyr11] somatostatin 14 as a radioligand. In 0.1 mM Ca2+ buffer the binding was saturable and slowly reversible, exhibiting a single class of high affinity binding sites (KD = 0.15 +/- 0.03 nM) with a maximal binding capacity (B max) of 178 +/- 18 fmol/mg protein. In 30 nM) free Ca2+ buffer, the binding was highly reversible. Affinity and B max were decreased by about 2-fold. Ca2+ exhibited an EC50 of 2.4 +/- 0.9 microM to potentiate the binding of somatostatin. Na+, but not K+, inhibited the binding: Bmax was decreased with no change in affinity. Somatostatin analogs inhibited the binding of 125I-[Tyr11] somatostatin 14. The relative potencies were: somatostatin 14 greater than somatostatin 28 = [Nle8]somatostatin 28 greater than [D Tryp8, D Cys14]somatostatin 14.     

Biphasic effect of protein kinase C on rat renal cortical Na+, K+-ATPase.

We examined the dependence of rat renal Na+, K+-ATPase activity on protein kinase C (PKC) stimulation. Infusion of either phorbol 12, 13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) into rat abdominal aorta resulted in dose-dependent changes of renal cortical Na+, K+-ATPase activity. Low doses of these esters (3 x 10(-11) mol/kg/min) increased activity of Na+, K+-ATPase whereas high doses (3 x 10(-9) mol/kg/min) decreased it. The changes in Na+, K+-ATPase activity induced by PDBu and PMA were prevented by staurosporine, a PKC inhibitor. 4Alpha phorbol didecanoate (4alpha PDD), phorbol ester which does not activate PKC had no effect on cortical Na+, K+-ATPase. PDBu and PMA did not change Na+, K+-ATPase activity in the renal medulla. The stimulatory effect of PDBu (3 x 10(-11) mol/kg/min) was neither mimicked by amphotericin B, a sodium ionophore nor blocked by amiloride, an inhibitor of Na+/H+-exchanger. The inhibitory effect of 3 x 10(-9) mol/kg/min PDBu was not mimicked by amiloride indicating that the observed effects of PKC stimulation are not secondary to alterations in intracellular sodium concentration. The inhibitory effect of PDBu was prevented by infusion of ethoxyresorufin, an inhibitor of cytochrome P450-dependent arachidonate metabolism. These results suggest that the inhibitory effect of PKC on renal cortical Na+, K+-ATPase is mediated by cytochrome P450-dependent arachidonate metabolites.     

Quinone and oxyradical scavenging properties of N-acetylcysteine prevent dopamine mediated inhibition of Na+, K+-ATPase and mitochondrial electron transport chain activity in rat brain: implications in the neuroprotective therapy of Parkinson's disease

Dopamine oxidation products such as H2O2 and reactive quinones have been held responsible for various toxic actions of dopamine, which have implications in the aetiopathogenesis of Parkinson's disease. This study has shown that N-acetylcysteine (0.25-1 mm) is a potent scavenger of both H2O2 and toxic quinones derived from dopamine and it further prevents dopamine mediated inhibition of Na+,K+-ATPase activity and mitochondrial respiratory chain function. The quinone scavenging ability of N-acetylcysteine is presumably related to its protective effect against dopamine mediated inhibition of mitochondrial respiratory chain activity. However, both H2O2 scavenging and quinone scavenging properties of N-acetylcysteine probably account for its protective effect against Na+,K+-ATPase inhibition induced by dopamine. The results have important implications in the neuroprotective therapy of sporadic Parkinson's disease since inactivation of mitochondrial respiratory activity and Na+,K+-ATPase may trigger intracellular damage pathways leading to the death of nigral dopaminergic neurons.     

Adenosine regulates the respiratory burst of cytokine-triggered human neutrophils adherent to biologic surfaces

The effect of adenosine on the respiratory burst was investigated using human neutrophils adherent to serum-coated surfaces. Adenosine caused complete suppression of the respiratory burst elicited by TNF-alpha, FMLP, or CSF for granulocytes; partial suppression of the response to CSF for granulocytes/macrophages, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, or uncoated polystyrene surfaces; and no suppression of the response to PMA. In most experiments, 4.7 x 10(-7) M and 2.5 x 10(-8) M adenosine caused 50% suppression of H2O2 release in response to TNF-alpha and FMLP, respectively, and 10 microM caused 100% suppression. Preexposure of neutrophils to ADP blocked the inhibitory effect of adenosine. With adherent neutrophils, there is a prolonged lag period in the onset of the respiratory burst in response to cytokines. Adenosine was fully suppressive if its addition was delayed past the first third of this lag period, or if it was removed during the last third of the lag period. A 10-min pulse with adenosine was most inhibitory when delivered in the middle third of the lag period. Dihydrocytochalasin B abolished the suppressive effect of adenosine on H2O2 release in response to FMLP. Thus adenosine, at concentrations found in human plasma, is a potent but selective inhibitor of the respiratory burst of adherent human neutrophils in response to physiologic, soluble stimuli, and ADP is a potentially physiologic counter-suppressant. Adenosine appears to exert most of its effect during a discrete interval within the lag period before onset of the respiratory burst, and may affect the coupling of agonist receptors to the cytoskeleton.     

Glutamatergically induced pattern of Ca2+ driving potential as a mechanism of postsynaptic plasticity.

Simulation studies were performed in a model of neuronal dendrite with Na+ and K+ channels and with ionotropic and metabotropic glutamate receptors. The ionotropic receptors were either N-methyl-D-aspartate (NMDA)-sensitive, voltage-dependent, and permeable to Ca2+, Na+, and K+, or non-NMDA-sensitive, voltage-independent, and permeable to Na+ and K+. The metabotropic receptors provided a catalytic effect on Ca2+-induced Ca2+ release from intracellular stores. Local intracellular concentration [Ca2+]i in the cytoplasm was changed because of exchange with the stores, axial diffusion, and transmembrane inward passive and outward pump fluxes. Tonic activation of ionotropic and metabotropic receptors in a particular range of intensities triggered the formation of spatially periodic [Ca2+]i hot and cold bands arising from an initial uniform state. The period and width of the bands were smaller at higher levels of tonic NMDA activation and higher metabotropically controlled rates of Ca2+-induced Ca2+ release. The bandwidths also depended on the dendrite diameter, the specific membrane, and cytoplasm resistivity. This activity-induced pattern led to long-term, spatially inhomogeneous change in local excitatory postsynaptic potentials (EPSPs) of NMDA synapses phasically activated with the same presynaptic intensity. The phasic EPSPs were potentiated if the synapse occurred in the hot band.