HORMONAL CONTROL OF LYSOSOMAL ENZYME RELEASE FROM HUMAN NEUTROPHILS : EFFECTS OF AUTONOMIC AGENTS ON ENZYME RELEASE, PHAGOCYTOSIS, AND CYCLIC NUCLEOTIDE LEVELS

The purpose of this investigation was to examine the effects of autonomic neurohormones, cyclic nucleotides, and related agents on the immunologic discharge of lysosomal enzymes from, and phagocytosis by, purified human neutrophils. In order to discern the possible intracellular mechanisms by which certain neurohormones influence neutrophil function, the concentrations of cyclic AMP and cyclic GMP in neutrophils were assessed during cell contact with phagocytizable particles and autonomic agents. The model system employed for study was the interaction of purified human neutrophils with rheumatoid arthritic (RA) serum-treated zymosan particles at 37°C in a neutral, balanced salt solution containing glucose. Neutrophils ingested the particles and discharged β-glucuronidase but not lactate dehydrogenase activity during 30 min of incubation. Treatment of zymosan particles with RA serum was more effective than treatment with normal serum with regard to the extent of both particle uptake and lysosomal enzyme release. During contact of neutrophils with RA serum-treated zymosan particles epinephrine, isoproterenol, and cyclic AMP inhibited both particle ingestion and β-glucuronidase discharge. These actions of epinephrine were associated with a concomitant elevation of cyclic AMP levels. In contrast to the actions of catecholamines and cyclic AMP, acetylcholine and cyclic GMP accelerated lysosomal enzyme release without affecting particle uptake. The actions of acetylcholine were associated with a concomitant elevation of cyclic GMP levels. Increases in neutrophil levels of cyclic GMP but not of cyclic AMP were associated also with the discharge of β-glucuronidase provoked by particles in the absence of added cholinergic agents. The data suggest that the immunologic release of lysosomal enzymes from human neutrophils can be regulated by autonomic neurohormones, perhaps via the selective formation of appropriate nucleotides.     

IMMUNOLOGICAL RELEASE OF HISTAMINE AND SLOW REACTING SUBSTANCE OF ANAPHYLAXIS FROM HUMAN LUNG : IV. ENHANCEMENT BY CHOLINERGIC AND ALPHA ADRENERGIC STIMULATION

The immunologic release of histamine and slow reacting substance of anaphylaxis (SRS-A) from human lung tissue can be enhanced by stimulation with either alpha adrenergic agents (phenylephrine or norepinephrine in the presence of propranolol) or cholinergic agents (acetylcholine or Carbachol). The finding that atropine prevents cholinergic but not comparable alpha adrenergic enhancement is consistent with the view that cholinergic and alpha adrenergic agonists interact with separate receptor sites on the target cells involved in the immunologic release of chemical mediators. The consistent qualitative relationship between the antigen-induced release of mediators and the level of cyclic adenosine monophosphate (cyclic AMP) as measured by the isolation of ¹⁴C-labeled cyclic AMP after incorporation of adenine-¹⁴C into the tissues or by the cyclic AMP binding protein assay suggests that changes in the level of this cyclic nucleotide mediate adrenergic modulation of the release of histamine and SRS-A. The addition of 8-bromo-cyclic guanosine monophosphate (cyclic GMP) produces an enhancement of the immunologic release of mediators while dibutyryl cyclic AMP is inhibitory. As cholinergic-induced enhancement was not associated with a measurable change in the levels of cyclic AMP, the possibility is suggested that cyclic GMP may be the intracellular mediator of cholinergic-induced enhancement of the immunologic release of histamine and SRS-A.     

Control and localization of rat adrenal cyclic guanosine 3'', 5''-monophosphate. Comparison with adrenal cyclic adenosine 3'', 5''-monophosphate.

Cyclic AMP and cyclic GMP were measured in rat adrenal glands after either hypophysectomy alone or after hypophysectomy and treatment with ACTH. Adrenal cyclic GMP levels rise in acutely hypophysectomized rats to a maximum at 1 h of approximately 200% of control levels; there is a return to base line at 4-12 h after hypophysectomy. In contrast, adrenal cyclic AMP falls immediately to about 50% of control levels after hypophysectomy and remains at approximately 1 pmol per mg tissue. Doses of ACTH beyond the physiological range markedly suppress adrenal cyclic GMP while producing a 50-fold or greater rise in cyclic AMP in hypophysectomized rats. This pattern of adrenal cyclic GMP rise was unchanged in acutely hypophysectomized animals treated with desamethasone. N-6-2'-0 dibutyryl cyclic AMP acted similarly to the effect of ACTH in bringing about a suppression of adrenal cyclic GMP levels. Physiological i.v. pulse doses of ACTH produced a rapid dose related increase in adrenal cyclic GMP. In vitro incubation of quartered adrenal pairs with 500 mU ACTH produced elevated cyclic AMP levels and suppression of cyclic GMP. Whereas adrenal cyclic AMP fell rapidly to 50% of control levels after hypophysectomy and remained at about 1 pmol per mg tissue for 7 days, adrenal cyclic GMP showed a biphasic rhythm in long-term hypophysectomized animals. After an initial peak at 1 h after hypophysectomy, adrenal cyclic GMP declined to baseline at 4-12 h but thereafter progressively rose with time, eventually reaching levels over 1 pmol per mg tissue. Fluorescent immunocytochemical staining of rat adrenal zona fasciculata showed cyclic AMP largely confined to cytoplasmic elements with little fluorescence contained in nuclei. In constant, cyclic GMP was found discretely positioned in nuclei with prominent fluorescence in nucleoli in addition to cytoplasmic localization. It is concluded that in hypophysectomized rats ACTH, either directly or in conjunction with altertion of adrenal cyclic AMP, appears to be one factor which regulates adrenal cyclic GMP. The direction of cyclic GMP change and the different subcellular localization of the nucleotides suggest divergent roles for cyclic AMP and cyclic GMP in adrenocortical function. Furthermore, our observations suggest a role for adrenal cyclic GMP in nuclear directed events.     

Increased levels of cyclic adenosine-3',5'-monophosphate in human polymorphonuclear leukocytes after surface stimulation.

Levels of cyclic AMP (cAMP) (but not cyclic GMP) in suspensions of human polymorphonuclear leukocytes (PMN) increased promptly after exposure of the cells to stimuli such as the chemotactic peptide N-formyl methionyl leucyl phenylalanine, the immune complex bovine serum albumin/anti-bovine serum albumin and calcium ionophore A23187. cAMP increased rapidly, reaching a maximum of twice the basal level 10--45 s after stimulation; after 2--5 min the amount of cAMP had subsided to basal levels. Elevations in cAMP levels were concurrent with, or followed, membrane hyperpolarization (measured by uptake of the lipophilic cation triphenylmethyl phosphonium) and always preceded lysosomal enzyme release and superoxide anion (O2) production. Elevated cAMP levels could be uncoupled from these later events by removal of extracellular divalent cations, replacement of extracellular Na+ with K+ or choline+, and by use of low concentrations of stimulus; each of these conditions virtually abolished lysosomal enzyme release and O2 generation, while leaving the stimulated elevation of cAMP levels unimpaired. Calcium ionophore A23187 did not provoke membrane hyperpolarization, thus uncoupling changes in membrane potential from changes in cAMP levels. These data suggested that cAMP is not a critical component in the earliest steps of stimulus-secretion coupling. Surface stimulation of cells pretreated with prostaglandins E1 or I2 yielded very high levels of cAMP; these high levels may be an important part of the mechanism by which stable prostaglandins inhibit lysosomal enzyme release and O2 generation.     

Effects of glucagon on adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate in human plasma and urine

Glucagon, infused intravenously into fasting, well-hydrated, normal men in doses of 25-200 ng/kg per min, induced up to 30-fold increases in both plasma and urinary cyclic AMP. Cyclic GMP levels were unaffected by glucagon. Simultaneous cyclic AMP and inulin clearance studies demonstrated that the glucagon-induced increase in urinary cyclic AMP was entirely due to glomerular filtration of the elevated plasma levels of the nucleotide.The cyclic AMP response to glucagon was not mediated by parathyroid hormone or epinephrine, and trypsintreated glucagon was completely inactive.The perfused rat liver released cyclic AMP into the perfusate in response to glucagon, indicating that the liver is a possible source of the cyclic AMP entering the extracellular fluids in response to glucagon in vivo.     

Differences in responsiveness of intrapulmonary artery and vein to arachidonic acid: mechanism of arterial relaxation involves cyclic guanosine 3':5'-monophosphate and cyclic adenosine 3':5'-monophosphate

The objective of this study was to examine the relationship between responses of bovine intrapulmonary artery and vein to arachidonic acid and cyclic nucleotide levels in order to better understand the mechanism of relaxation elicited by arachidonic acid and acetylcholine. Arachidonic acid relaxed phenylephrine-precontracted arterial rings and elevated both cyclic GMP and cyclic AMP levels in arteries with intact endothelium. In contrast, endothelium-damaged arterial rings contracted to arachidonic acid without demonstrating significant changes in cyclic nucleotide levels. Indomethacin partially inhibited endothelium-dependent relaxation and abolished cyclic AMP accumulation whereas methylene blue, a guanylate cyclase inhibitor, partially inhibited relaxation and abolished cyclic GMP accumulation in response to arachidonic acid. All vessel responses were blocked by a combination of the two inhibitors. Prostaglandin (PG) I2 relaxed arterial rings and elevated cyclic AMP levels whereas PGE2 and PGF2 alpha caused contraction, suggesting that the indomethacin-sensitive component of arachidonic acid-elicited relaxation is due to PGI2 formation and cyclic AMP accumulation. The methylene blue-sensitive component is attributed to an endothelium-dependent but cyclooxygenase-independent generation of a substance causing cyclic GMP accumulation. Intrapulmonary veins contracted to arachidonic acid with no changes in cyclic nucleotide levels and PGI2 was without effect. Homogenates of intrapulmonary artery and vein formed 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from [14C]arachidonic acid, which was inhibited by indomethacin. Thus, bovine intrapulmonary vein may not possess receptors for PGI2. The failure of endothelium-intact vein to relax to acetylcholine may be related to the lack of a relaxant effect by arachidonic acid, perhaps attributed to the absence of generation of an endothelium-derived relaxing factor.     

Decreased H2 Histamine Response of Granulocytes of Asthmatic Patients

Increased bronchial sensitivity to inhaled histamine in asthma is well known. The mechanism of this increased bronchial sensitivity is not known nor has it been demonstrated that isolated cells respond abnormally to histamine. Polymorpho-nuclear leukocytes (PMNs) provide a homogeneous cell population to study agonist response. Release of granulocyte lysosomal enzymes is inhibited by agonists increasing the PMN cyclic AMP concentration. The release of the lysosomal enzyme beta glucuronidase by serum-activated particles of zymosan was similar in PMNs isolated from normal and asthma subjects. Histamine (100-0.01 muM) inhibited enzyme release. Except at the maximal concentration of histamine (100 muM), the response to histamine was decreased in asthma. The inhibition of enzyme release paralleled an increase in intracellular PMN cyclic AMP. In asthma, the cyclic AMP response to histamine was reduced. The H2 antihistamine metiamide blocked histamine inhibition of lysosomal enzyme release and the increase in cyclic AMP. The effect was maximal at concentrations equimolar to those of histamine. The H1 antihistamine chlorpheniramine had no effect on histamine inhibition of granulocyte lysosomal enzyme release. A decrease in the inhibition of the release of the inflammatory lysosomal enzymes from granulocytes in asthma may contribute to an enhanced bronchial inflammatory reaction.     

LASTING BIOLOGICAL EFFECTS OF EARLY ENVIRONMENTAL INFLUENCES : VII. METABOLISM OF ADENOSINE 3'',5''-MONOPHOSPHATE IN MICE EXPOSED TO EARLY ENVIRONMENTAL STRESS

The metabolism of adenosine 3',5'-monophosphate (cyclic AMP) was studied in specific pathogen-free mice exposed to neonatal infection with mouse enterovirus or to malnutrition during early life. Metabolic activity was determined by measuring the turnover of cyclic AMP-8-¹⁴C to respiratory ¹⁴CO₂, its incorporation into various organs and plasma, and the binding activity of synaptosome for cyclic AMP. Early malnutrition increased the catabolism of cyclic AMP as measured by expiration in respiratory CO₂. The level of cyclic AMP was lower in plasma and its incorporation into various tissues was decreased in infected and malnourished animals. Metabolic products of cyclic AMP were isolated from plasma by ion exchange chromatography. Cyclic AMP-8-¹⁴C had completely disappeared 9 hr after injection. Fewer metabolites of cyclic AMP were detected in infected or malnourished groups than in controls and the metabolic reaction from 5'-AMP to adenosine seemed to be slow in these animals. The ability to incorporate cyclic AMP to synaptosome was also impaired in the experimental groups. The concentrations of brain cyclic AMP were lower in infected or malnourished animals than in controls. Depression of accumulation of cyclic AMP probably resulted from excessive activity of phosphodiesterase, rather than from impairment of adenyl cyclase. Intraperitoneal administration of theophylline brought the activity level of phosphodiesterase to normal in infected or malnourished mice; this fact probably accounted for enhanced accumulation of brain cyclic AMP.     

Diminished Polymorphonuclear Leukocyte Adherence: FUNCTION DEPENDENT ON RELEASE OF CYCLIC AMP BY ENDOTHELIAL CELLS AFTER STIMULATION OF β-RECEPTORS BY EPINEPHRINE

To investigate the biochemical and cellular basis for the rise in polymorphonuclear leukocyte (PMN) count during epinephrine administration, PMN from subjects receiving epinephrine were studied for their capacity to adhere to nylon wool fibers and endothelial cell monolayers. After administration of epinephrine, the PMN count increased by 80% at 5 min, and isolated PMN adherence to nylon fibers fell from a base line of 44±2-18±3%. In contrast, when subjects were infused with the β-antagonist propanolol before receiving epinephrine, the PMN count failed to rise and PMN adherence was normal. Exposure of PMN endothelial cell monolayers to 0.1 μM epinephrine led to diminished PMN adherence that could be blocked by 10 μM propanolol but not by 10 μM phentolamine. Sera obtained from subjects 5 min after receiving epinephrine or from supernates derived from endothelial cell monolayers exposed to 90 nM epinephrine inhibited PMN adherence to nylon fibers. Addition of anticyclic AMP antisera but not anticyclic guanosine monophosphate antisera to the postepinephrine sera or to the postepinephrine supernate derived from the endothelial cell monolayers abolished their inhibitory effect of PMN adherence to nylon fibers. In contrast, direct exposure of PMN to epinephrine failed to affect their adherent properties. Because it has been previously shown that endothelial cells contain β-receptors and respond to catecholamines by raising their intracellular concentrations of cyclic AMP, and that PMN adherence is attenuated by cyclic AMP, it would appear that diminished PMN adherence after epinephrine administration is mediated through endothelial cell β-receptor activity, which in turn impairs PMN margination in vivo and could account for the rise in circulating PMN.     

Cyclic 3′,5′-adenosine monophosphate in human blood platelets: II. Effect of N6-2′-0-dibutyryl cyclic 3′,5′-adenosine monophosphate on platelet function

The relation of cyclic 3',5'-adenosine monophosphate to platelet function has been studied by investigating the influence of this compound and of its N(6)-2'-0-dibutyryl derivative on platelet aggregation and other aspects of platelet behavior after demonstration of adenyl cyclase activity in disrupted platelets.Dibutyryl cyclic AMP inhibited platelet aggregation induced by ADP, epinephrine, collagen, and thrombin. Cyclic AMP was also inhibitory but was less effective. The platelet "release reaction" was also inhibited; specifically, there was inhibition of the induction of platelet factor 3 activity and of the release of labeled 5-hydroxytryptamine. Platelet swelling produced by ADP was not inhibited.The action of dibutyryl cyclic AMP did not result from contamination with 5'-AMP, nor was it attributable to production of 5'-AMP by plasma enzymes. Dibutyryl cyclic AMP was degraded to 2'-O-monobutyryl cyclic AMP and to cyclic AMP in plasma, but plasma exhibited no cyclic nucleotide phosphodiesterase activity, and the production of 5'-AMP did not occur. The in vitro effects of dibutyryl cyclic AMP were associated with uptake of the compound by platelets.Adenyl cyclase activity of platelet homogenates was demonstrated with production of 9.27 x 10(-11) (+/-2.62 x 10(-11)) mole cyclic AMP per min per 10(10) platelets. The activity was increased by NaF and by prostaglandin PGE(1) and was decreased by epinephrine. The effect of epinephrine was blocked by phentolamine but not by propanolol. Adenyl cyclase activity was also inhibited by collagen, 5-hydroxytryptamine, and thrombin. ADP, dibutyryl cyclic AMP, and cyclic AMP did not alter adenyl cyclase activity.These observations are consistent with the hypothesis that platelet aggregation is favored by a decrease in platelet cyclic AMP and inhibited by an increase in cyclic AMP.     

Human Lung Tissue and Anaphylaxis: EVIDENCE THAT CYCLIC NUCLEOTIDES MODULATE THE IMMUNOLOGIC RELEASE OF MEDIATORS THROUGH EFFECTS ON MICROTUBULAR ASSEMBLY

The addition of specific antigen to IgE-sensitized human lung tissue causes the secretion of the mediators histamine and slow-reacting substance of anaphylaxis. The mechanisms by which increased levels of cyclic AMP suppress and increased levels of cyclic GMP enhance this secretory process were studied. Colchicine, an agent which disrupts many secretory reactions by binding to microtubules in their disassembled 6S form, was a relatively ineffective inhibitor of the antigen-induced release of mediators unless lung fragments were incubated at 4 degrees C for 60 min to induce microtubular disassembly. As colchicine appeared to inhibit the immunologic secretion of mediators from human lung tissue most effectively after microtubular disassembly, the capacity of colchicine to modulate the release reaction indicated the state of microtubular assembly; inhibition by colchicine signaled a shift to the colchicine-sensitive 6S subunits whereas failure to inhibit suggested maintenance in the colchicine-resistant polymerized state.Exogenously added 8-Bromo-cyclic GMP prevented low temperature-facilitated colchicine suppression of mediator release suggesting that increased levels of cyclic GMP stabilize polymerized microtubules. Transiently increased cyclic AMP concentrations, either exogenously added as 8-Bromo-cyclic AMP or endogenously produced by isoproterenol, promoted colchicine suppression of mediator release suggesting that microtubular disassembly was produced. Direct measurement of cyclic AMP levels revealed parallel kinetics after isoproterenol stimulation between control and colchicine-treated lung fragments.The requirement for functional microtubules in the release reaction may occur after the antigen IgE-stimulated activation of a serine esterase, energy utilization, and an intracellular calcium requirement. The mechanism by which cyclic nucleotides influence microtubular assembly is postulated to involve the degree of phosphorylation-dephosphorylation of microtubules.     

Effects of Cholera Enterotoxin on Adenosine 3′,5′-Monophosphate and Neutrophil Function. COMPARISON WITH OTHER COMPOUNDS WHICH STIMULATE LEUKOCYTE ADENYL CYCLASE

Cholera enterotoxin caused a delayed accumulation of adenosine 3',5'-monophosphate (cyclic AMP) in human leukocytes, associated with an increase in leukocyte adenyl cyclase activity. The action of cholera enterotoxin contrasted with that of other agents which stimulate adenyl cyclase: (a) the effects of the toxin were delayed in onset, while prostaglandin-E(1) (PGE(1)) and isoproterenol acted rapidly; (b) removal of the soluble toxin from the extracellular medium did not abolish its effects on cyclic AMP and inhibition of antigenic histamine release, while removal of PGE(1) did prevent its effects; (c) PGE(1), but not cholera enterotoxin, stimulated adenyl cyclase activity when added directly to broken cell preparations. Binding of the toxin to leukocytes was rapid and irreversible, and was followed by a gradual increase in cyclic AMP which was not prevented by cycloheximide.Cholera enterotoxin caused accumulation of cyclic AMP in purified human neutrophils as well as mono-nuclear cells, but did not prevent the extrusion of lysosomal hydrolases from phagocytic cells. The toxin only slightly inhibited the ability of human neutrophils to kill Candida albicans. Thus these results with the toxin cast doubt on previous proposals that cyclic AMP regulates these two functions of neutrophils. The unique action of cholera enterotoxin on cyclic AMP production provides a potentially useful pharmacologic tool, in addition to methylxanthines and dibutyryl cyclic AMP, for testing hypotheses relating cyclic AMP to altered function of leukocytes and, perhaps, of other mammalian cells.     

ACTIVATION OF LYSOSOMAL ENZYMES IN VIRUS-INFECTED CELLS AND ITS POSSIBLE RELATIONSHIP TO CYTOPATHIC EFFECTS

Activities of the enzymes β-glucuronidase, acid phosphatase, acid DNAase, acid RNAase, and acid protease have been measured in the lysosomal and supernatant fractions of mouse liver cells and monkey kidney cells before and after infection with mouse hepatitis virus and vaccinia virus, respectively. In the infected cells there was easily measurable release of lysosomal enzymes into the supernatant fraction. Evidence was presented that this is not an artefact of homogenization and precedes cell degeneration demonstrable histologically. It is suggested that release of lysosomal enzymes may explain some of the biochemical changes found in infected cells and may contribute to the cytopathic effects of some viruses.     

Muscarinic cholinergic regulation of cyclic guanosine 3,5-monophosphate in autonomic ganglia: possible role in synaptic transmission.

The effects of acetylcholine, and of agents which selectively mimic or block its nicotinic and muscarinic actions, have been studied on the levels of guanosine 3,5-monophosphate (cyclic GMP) and adenosine 3,5-monophosphate (cyclic AMP) in slices of bovine superior cervical ganglion. Low doses of either acetylcholine or the muscarinic agonist, bethanechol, caused a substantial increase in the level of cyclic GMP, and slightly increased the level of cyclic AMP; the acetylcholine- and the bethanechol-induced increases in cyclic GMP were blocked by atropine, the muscarinic antagonist, but not by hexamethonium, the nicotinic antagonist. The nicotinic agonist N,N-dimethylphenylpiperazinium did not alter the level of either of the cyclic nucleotides. Dopamine increased the level of cyclic AMP and acetylcholine partially prevented this dopamine-induced increase in cyclic AMP. The data are consistent with a model for the ganglion in which muscarinic cholinergic synaptic transmission is mediated by an increase in the level of cyclic GMP in the postganglionic neurons and dopaminergic transmission by an increase in the level of cyclic AMP in these neurons.     

Beta-adrenergic regulation of cyclic adenosine 3'',5'' monophosphate accumulation in human gastric epithelial glands. Inhibitory effect of somatostatin

The action of catecholamines and somatostatin on cyclic adenosine 3',5' monophosphate (cyclic AMP) formation in human isolated gastric glands is reported. We show that: (1) there is a beta 2 receptor-mediated stimulation of cyclic AMP production in fundus. Catecholamines act with the order of potencies isoproterenol (ED50 = 50 nmol 1(-1) greater than epinephrine (ED50 = 0.1 mumol 1(-1] greater than norepinephrine (ED50 = 5 mumol 1(-1]. Their action is completely reversed by propranolol at doses 10(3) times lower than practolol, while unaffected by phentolamine; (2) isoproterenol and Vasoactive Intestinal Peptide (VIP) have additive effects on cyclic AMP in fundic glands whereas no additivity is observed between histamine and isoproterenol; this, together with the absence of catecholamine effect in antral glands, suggests that the beta 2 receptor is located on parietal cells; (3) somatostatin (1 mumol 1(-1] non-competitively inhibits the stimulation by catecholamines but does not affect VIP and histamine stimulations. These results suggest a physiological stimulatory effect of catecholamines on gastric acid secretion in man, through a beta 2 receptor coupled to the cyclic AMP system, regulated by somatostatin.     

RELEASE OF CARTILAGE MUCOPOLYSACCHARIDE-DEGRADING NEUTRAL PROTEASE FROM HUMAN LEUKOCYTES

The granule fraction of human leukocytes contains neutral protease capable of degrading the noncollagenous protein mucopolysaccharide matrix of cartilage at neutral pH in physiological salt solution. Cartilage degradation was monitored by quantitating the release of ³⁵S from labeled rabbit ear cartilage. Degradation of cartilage matrix occurs when intact viable human leukocytes are incubated with cartilage opsonized with aggregated human gamma globulin (AHGG). During a similar 4 h incubation period cells did not degrade uncoated cartilage or cartilage coated with nonaggregated gamma globulin. Cells remain viable during the enzyme release process as evidenced by the absence of a cytoplasmic enzyme marker (lactic dehydrogenase) in the supernatant and dye exclusion studies. The release of ³⁵S from labeled cartilage by human leukocytes in the presence of cartilage coated with AHGG (nonphagocytic enzyme release) was compared with the cartilage degrading activity of the supernatant from the same number of cells preincubated with a suspension of AHGG (phagocytic enzyme release). Nonphagocytic enzyme release by 5 x 10⁶ cells provoked two to four times more ³⁵S and β-glucuronidase (β-G) release from cartilage than phagocytic enzyme release conditions. β-glucuronidase was used as an indicator of the release of lysosomal granule enzymes. By the use of selected pharmacological agents it was possible to dissociate the enzyme release process from intrinsic enzyme (neutral protease) activity. Neutral protease and β-G release by human cells in the presence of AHGG-coated cartilage was inhibited by 10^–5M colchicine, whereas the protease activity, but not the release process, was inhibited by 10^–6M gold thiomalate and 10% human serum. It is suggested that the release of a cartilage degrading neutral protease by viable human cells when exposed to AHGG might be a relevant model for the study of cartilage destruction as it occurs in rheumatoid arthritis.     

Effects of catecholamines and adrenergic-blocking agents on plasma and urinary cyclic nucleotides in man

Studies were performed in healthy volunteers to determine the effects of catecholamines and adrenergic-blocking agents on plasma and urinary levels of adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP).Plasma cyclic AMP rose in response to infusions of the beta-adrenergic agent, isoproterenol, or in response to infusions of either epinephrine or norepinephrine alone or in combination with the alpha-adrenergic-blocking agent, phentolamine. Although urinary cyclic AMP also rose, the percentage increase was less than that observed in the plasma. These treatments caused no increase in plasma cyclic GMP.Plasma cyclic GMP rose in response to infusions of alpha-adrenergic agents, viz., epinephrine or norepinephrine infused together with the beta-blocking agent, propranolol. These treatments caused no increase in plasma cyclic AMP.These observations are consistent with the current concept that the actions of beta-adrenergic agents are mediated by increases in cyclic AMP formation in target tissues. Such a mediating role has not been established for cyclic GMP, but the data suggest the possibility that cyclic GMP metabolism is responsive either to alpha-adrenergic stimulation or to parasympathetic stimulation which occurs as a reflexive consequence of the pressor effect of alpha-adrenergic agents.     

Regulation of cyclic nucleotide levels and glycogen phosphorylase activity by acetylcholine and epinephrine in perfused rat hearts.

Acetylcholine (ACh) produced a time and dose-dependent increase in cyclic 3',5'-guanosine monophosphate (cyclic GMP) levels in perfused rat hearts without any significant (P greater than .05) change in cyclic 3',5'-adensoine monophosphate (cyclic AMP) level or glycogen phosphorylase activity. Epinephrine produced a dose-dependent increase in both cyclin AMP and phosphorylase activity but no significant (P greater than .05) change in cyclic GMP levels. When ACh (10(-6) M) was infused into hearts during the infusion of epinephrine (10(-6) M), a time-dependent increase in cyclic GMP and decrease in cyclic AMP occurred, both of which preceded a significant (P less than .05) decrease in glycogen phosphorylase activity. In epinephrine-stimulated hearts, ACh produced a dose-dependent increase in cyclic GMP levels. Similar concentrations produced a fall in cyclic AMP levels and phosphorylase activity. No condition tested resulted in a significant (P greater than .05) change in glycogen synthase activity. It is concluded that ACh can reduce cyclic AMP levels and phosphorylase activity only when they have been elevated above basal values. These changes are associated with an increase in cyclic GMP. The reduction in phosphorylase produced by ACh may be the result of either or both of the changes in cyclic nucleotide levels.     

Kinetic parameters and renal clearances of plasma adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate in man

Kinetic parameters and the renal clearances of plasma adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP) were evaluated in normal subjects using tritium-labeled cyclic nucleotides. Each tracer was administered both by single, rapid intravenous injection and by constant intravenous infusion, and the specific activities of the cyclic nucleotides in plasma and urine were determined.Both cyclic AMP and cyclic GMP were cleared from plasma by glomerular filtration. The kidney was found to add a variable quantity of endogenous cyclic AMP to the tubular urine, amounting to an average of approximately one-third of the total level of cyclic AMP excreted. Plasma was the source of virtually all of the cyclic GMP excreted.Plasma levels of the cyclic nucleotides appeared to be in dynamic steady state. The apparent volumes of distribution of both nucleotides exceeded extracellular fluid volume, averaging 27 and 38% of body weight for cyclic AMP and cyclic GMP, respectively. Plasma production rates ranged from 9 to 17 nmoles/min for cyclic AMP and from 7 to 13 nmoles/min for cyclic GMP. Plasma clearance rates averaged 668 ml/min for cyclic AMP and 855 ml/min for cyclic GMP. Approximately 85% of the elimination of the cyclic nucleotides from the circulation was due to extrarenal clearance.     

Studies on the activities, kinetic properties and subcellular localisation of cyclic AMP phosphodiesterase in human neutrophil leukocytes

A one-step radioassay for cyclic AMP phosphodiesterase was optimised for human polymorphonuclear leukocytes. Kinetic studies indicated the presence of two forms of phosphodiesterase activity with apparent Km values of 0.015 mmol/l and 0.98 mmol/l for cyclic AMP. Control neutrophils were homogenised in isotonic sucrose and, after low speed centrifugation, the supernatant was subjected to analytical subcellular fractionation. Gradient fractions were assayed for principal marker enzymes and for cyclic AMP phosphodiesterase. Both forms of phosphodiesterase activity were located in the cytosol. Polymorphonuclear leukocyte homogenates were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. A portion of each homogenate was used for enzyme analysis and the remainder assayed for cyclic AMP content. The specific activity (mUnits/mg protein) of the low Km phosphodiesterase was reduced in both patient groups compared with control values, whilst that of the high Km phosphodiesterase was unchanged. Leukocytes from patients with chronic granulocytic leukaemia had only a fifth of the cyclic AMP content of control neutrophils, whilst leukocytes from patients in the third trimester of pregnancy had an elevated cyclic AMP content.