An isotopic assay for NADPH oxidase activity and some characteristics of the enzyme from human polymorphonuclear leukocytes.

An isotopic assay for NADPH ixodase that measures the amount of NADP formed by the 6-phosphogluconate dehydrogenase reaction has been developed. Under appropriate conditions, the amount of NADP present is directly proportional to the amount of 14CO2 released from [1-14C]6-phosphogluconic acid. Because this assay employs radioisotopes, it is far more sensitive than conventional assays for the enzyme. The human granule NADPH oxidase, as measured by this assay, is active in the presence of CN minus, is stimulated by Mn-2+, and has a pth optimum of 5.5. Granules isolated from cells that have been allowed to ingest zymosan consistently exhibited more enzyme activity than did granules isolated from either resting cells or cells challenged with zymosan that was not preopsonized. This effect was observed over a wide range of substrate concentrations and could not be explained by differences in protein concentrations between the various samples. If whole homogenates are used in place of isolated granules, the enzyme activity can be observed only with a homogenate of phagocytizing cells and even then only at a high concentration of NADPH. This suggests that an inhibitor of the enzyme might be present within the cell. One patient with chronic granulomatous disease was studied. There was no difference in tnadph oxidase activity of the patients' cells when granules from resting and phagocytizing cells were compared. In contrast, the enzyme activity in granules from two control patients doubled upon phagocytosis. These results are consistent with a role for NADPH oxidase in the initiation of the respiratory burst accompanying phagocytosis by human neutrophils.

Identification of alpha-adrenergic receptors in uterine smooth muscle membranes by [3H]dihydroergocryptine binding.

[3H]Dihydroergocryptine, a potent alpha-adrenergic antagonist, was used to label smooth muscle membrane binding sites which have the characteristics expected of alpha-adrenergic receptors. Binding of [3H]dihydroergocryptine to rabbit uterine membranes was rapid and reversible with rate constants of 1.26 X 10(7) M-1 min-1 and 0.034 min-1 for the forward and reverse reactions, respectively. [3H]Dihydroergocryptine binding was of high affinity, with an equilibrium dissociation constant (KD) of 8 to 10 nM. Binding was saturable with 0.14 to 0.17 pmol of [3H]dihydroergocryptine bound/mg of protein at maximal occupancy of the sites. No cooperative interactions among the sites were detected. The specificity of the binding sites for a large number of adrenergic agonists and antagonists was identical with the specificity of alpha-adrenergic responses to these agents. The alpha-adrenergic agonist (-)-epinephrine competed for binding with a KD of 0.23 muM. The order of potencies for several adrenergic agonists in competing for the binding sites was (-)-epinephrine greater than (-)-norepinephrine greater than (-)-phenylephrine greater than (-)-isoproterenol in agreement with their alpha-adrenergic potencies. A series of 19 phenylethylamine adrenergic agonists competed for binding in a manner paralleling their potencies as alpha-adrenergic agonists. alpha-Adrenergic antagonists such as phentolamine (KD = 15 nM) and phenoxybenzamine (KD = 18 nM) potently competed for the binding sites. In contrast, beta-adrenergic antagonists such as propranolol (KD = 27,000 nM) and practolol (KD greater than 10(6) nM) did not have high affinity for the binding sites. A series of ergot alkaloids competed for [3H]dihydroergocryptine binding in a manner which paralleled their potencies as alpha-adrenergic agents. Competition for binding sites by alpha-adrenergic agonists and antagonists was a stereospecific process. The (-)-stereoi somers of epinephrine, norepinephrine, and ergotamine were at least 20- to 50-fold more potent than the corresponding (+)-stereoisomers. Compounds devoid of significant alpha-adrenergic activity, such as pyrocatechol, 3,4-dihydroxymandelic acid, normetanephrine, and D-lysergic acid, did not effectively compete for [3H]dihydroergocryptine binding sites. These rabbit uterine binding sites for [3H]dihydroergocryptine appear to have characteristics indistinguishable from those of the physiologically active alpha-adrenergic receptors.

Regulation of beta-adrenergic receptors by guanyl-5'-yl imidodiphosphate and other purine nucleotides.

Guanyl-5'-yl imidodiphosphate (Gpp(NH)p), GTP, and other purine nucleotides selectively decrease the binding affinity of the beta-adrenergic receptors of frog erythrocyte membranes for beta-adrenergic agonists but not antagonists. Shifts in binding affinity were assessed by determining the ability of unlabeled ligands to compete with (-)-[3H]dihydroalprenolol for the membrane-bound receptors. The magnitude of the"right" shift in the binding displacement curve for any of 13 ligands tested was directly related to the intrinsic activity (maximal stimulatory capacity) of that agent for stimulation of the frog erythrocyte membrane adenylate cyclase. Thus, Gpp(NH)p-induced shifts in binding affinity were greatest for full agonists such as isoproterenol, intermediate for partial agonists such as soterenol, and no shifts were observed for antagonists such as propranolol. Shifts in binding affinity were observed only in preparations where agonist binding to the receptors leads to "coupling" of the receptors with adenylate cyclase. In solubilized preparations where the beta-adrenergic receptors and adenylate cyclase are functionally "uncoupled", Gpp(NH)p did not cause right shifts in agonist receptor binding displacement curves. In particulate preparations the Km of Gpp(NH)p for stimulation of adenylate cyclase was identical with that for its effect on beta-adrenergic agonist binding affinity, 1 to 2 muM. Moreover, the ability of several other nucleotides to cause shifts in receptor binding affinity directly paralleled their previously determined affinities for the nucleotide regulatory sites on adenylate cyclase. Gpp(NH)p also shifted agonist dose-response curves for stimulation of adenylate cyclase, but to the left. As with the effects on the receptor binding curves, the effects of Gpp(NH)p on the "apparent affinities" of agonists for enzyme stimulation were directly related to their intrinsic activities. Gpp(NH)p also markedly increased the intrinsic activity of partial agonists. These results appear to indicate that conformational alterations in adenylate cyclase caused by occupation of nucleotide regulatory sites by Gpp(NH)p are capable of inducing alterations in the beta-adrenergic receptors. These receptor alterations are induced only when the receptors are "coupled" to the enzyme by virtue of agonist binding. The nucleotide-altered conformation of the beta-adrenergic receptors is characterized by decreased binding affinity for agonist but increased functional efficacy in stimulating the enzyme.

Differences between agonist and antagonist binding following beta-adrenergic receptor desensitization.

The specific beta-adrenergic agonist radioligand (+/-)-[3H]hydroxybenzylisoproterenol ([3H]HBI) was used to investigate alterations in the beta-adrenergic receptors of frog erythrocytes occurring during the process of agonist-induced, receptor-specific desensitization. There was close agreement between the percentage fall in [3H]HBI binding and that in catecholamine-stimulated adenylate cyclase activity following periods of preincubation of up to 7 h with 0.1 mM (-)-isoproterenol. Desensitization was maximal by 5 h, resulting in a 69% reduction in [3H]HBI binding and a 67% reduction in isoproterenol-stimulated adenylate cyclase activity. In contrast, binding of the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol was significantly less affected by desensitization (p is less than 0.05 at 2 1/2, 5, and 7 h), showing a maximum reduction in binding of only 35% in these experiments. The consistent close agreement of reduction in agonist binding with that in hormone-stimulated adenylate cyclase activity, together with the significant difference observed between agonist and antagonist binding, implies that an alteration occurs during desensitization which preferentially interferes with agonist binding, while antagonist binding is less affected. The locus of this agonist-specific alteration may be the receptor binding site or a site involved in receptor-enzyme coupling. Agonist binding studies may now be used to assess more completely the desensitized state of beta-adrenergic receptors in systems in which marked desensitization of beta-adrenergic responses is associated with little or no reduction in antagonist binding.

Phagocytosis of live versus heat-killed bacteria by human polymorphonuclear leukocytes.

Heat-killed Pseudomonas aeruginosa are phagocytized much more slowly by human polymorphonuclear leukocytes than are the live organisms. The post-phagocytic increase in hexose monophosphate shunt activity (HMS) parallels the ingestion of the bacteria. The addition of serum to the live organisms causes a marked increase in both ingestion and cellular HMS activity; serum actually causes an inhibition of both uptake and HMS activity when added to the heat-killed organisms. Differences in postphagocytic HMS activity between live and heat-killed organisms were observed with three different species of bacteria, indicating that the phenomenon is not restricted to P. aeruginosa. These data emphasize that the influence of the particle on the phagocytic process is considerable.

Reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate diaphorase activity in human polymorphonuclear leukocytes.

Total reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activities were examined in human neutrophils. Approximately two-thirds of each enzyme activity was located in the granule fraction with the remainder in the soluble. The activities in a 27,000 x g supernatant from a sonic extract of human polymorphonuclear leukocytes were characterized. Both NADH and NADPH diaphorase were insensitive to cyanide and azide and showed greater activity at acid pH. K(m) values for nitroblue tetrazolium were not markedly different (33 muM with NADH and 12 muM with NADPH), but there was a 40-fold difference in K(m) for the reduced pyridine nucleotides (10 muM with NADH and 400 muM for NADPH). Since the intracellular concentration of both nucleotides is estimated to be about 50 muM, it is much more likely, from a kinetic argument, that the respiratory burst of phagocytosis is intiated by the oxidation of NADH rather than of NADPH.