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The mechanism of activation of soluble guanylate cyclase purified from bovine lung by high molecular weight, nitrosyl-hemoprotein complexes is reported. Heme-containing, heme-deficient, and heme-reconstituted forms of guanylate cyclase were studied. Nitric oxide (NO) and nitroso compounds activated heme-containing and heme-reconstituted enzymes (over 50-fold), with an accompanying shift in the Soret absorption peak from 431 to 398 nm, but failed to activate or alter the spectral characteristics of heme-deficient enzyme. In contrast, preformed NO-hemoprotein complexes as well as low molecular weight NO-heme activated all forms of guanylate cyclase. Heme-deficient guanylate cyclase was first reacted with excess amounts of NO-hemoglobin, NO-myoglobin, or NO-catalase and then rapidly separated from the NO-hemoprotein by column chromatography. Spectrophotometric analysis indicated that the NO-heme moiety was transferred from each of the NO-hemoproteins to heme-deficient guanylate cyclase. Approximately 1 mol of NO-heme was bound per mol of holoenzyme and the specific activity of this enzyme form was over 50-fold greater than that of unreacted, heme-deficient enzyme. NO-heme was tightly bound to guanylate cyclase as no transfer of enzyme-bound NO-heme to apohemoglobin was evident. Enzyme activated by NO-hemoproteins closely resembled, kinetically, that activated by NO or NO-heme. In contrast, reactions between heme-deficient guanylate cyclase and hemoproteins did not result in heme transfer, whereas heme alone rapidly reconstituted the enzyme. These observations indicate that soluble guanylate cyclase can be readily reconstituted with, and thereby activated by, NO-heme through an exchange reaction with NO-hemoproteins.

The biologic role of calcium and guanosine 3':5'-monophosphate (cyclic GMP) in the immunologic secretion of lysosomal enzymes from human neutrophils was studied. Contact of neutrophils with zymosan-treated serum or the divalent cation ionophore A-23187, in the presence of extracellular calcium, resulted in beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) secretion and a concomitant accumulation of cyclic GMP without any loss of cell viability. Acetylcholine (0.1 muM), in the presence of calcium, enhanced the immunologic stimulation of cyclic GMP accumulation and lysosomal enzyme discharge. A marked and rapid association of 45CaCl2 with neutrophils occurred during cell surface contact with zymosan-treated serum, and this effect on calcium association was enhanced with 0.1 muM acetylcholine. The precise mechanism of the neutrophil-calcium interaction is presently not well understood. However, the finding that 0.5-1.0 muM A-23187 also provoked a rapid association of extracellular calcium with neutrophils suggests that calcium mobilization into the intracellular environment may account, at least in part, for this association between cells and calcium. The close temporal relationship between beta-glucuronidase secretion, cyclic GMP accumulation, and calcium mobilization during cell contact with membrane active agents such as immune reactants, acetylcholine, and ionophores suggests that these three cellular events bear a cause and effect relationship. On the basis of our findings to date, we propose that the immunologic secretion of lysosomal contents from human neutrophils is signaled by intracellular cyclic GMP and that extracellular calcium, by gaining access to the intracellular processes responsible for cyclic GMP accumulation, serves as the link to stimulus-secretion coupling.

The principal objective of this study was to test the hypothesis that nitroprusside relaxes vascular smooth muscle via the reactive intermediate, nitric oxide (NO), and that the biologic action of NO is associated with the activation of guanylate cyclase. Nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and NO elicit concentration-dependent relaxation of precontraced helical strips of bovine coronary artery. Nitroprusside, MNNG and NO also markedly activate soluble guanylate cyclase from bovine coronary arterial smooth muscle and, thereby, stimulate the formation of cyclic GMP. Three heme proteins, hemoglobin, methemoglobin and myoglobin, and the oxidant, methylene blue, abolish the coronary arterial relaxation elicited by NO. Similarly, these heme proteins, methylene blue and another oxidant, ferricyanide, markedly inhibit the activation of coronary arterial guanylate cyclase by NO, nitroprusside and MNNG. The following findings support the view that certain nitroso-containing compounds liberate NO in tissue:heme proteins, which cannot permeate cells, inhibit coronary arterial relaxation elicited by NO, but not by nitroprusside or MNNG; the vital stain, methylene blue, inhibits relaxation by NO, nitroprusside and MNNG; heme proteins and oxidants inhibit guanylate cyclase activation by NO, nitroprusside and MNNG in cell-free mixtures. The findings that inhibitors of NO-induced relaxation of coronary artery also inhibit coronary arterial guanylate cyclase activation suggest that cyclic GMP formation may be associated with coronary arterial smooth muscle relaxation.

The principal objective of this study was to compare and contrast the vascular smooth muscle-relaxing properties of endothelium-derived relaxing factor (EDRF) and nitric oxide (NO) in two different assay systems. In one system, precontracted rings of bovine intrapulmonary artery and vein and coronary artery were used to compare the relaxant effects of endothelium-dependent vasodilators, NO, S-nitroso-N-acetylpenicillamine, glyceryl trinitrate, prostacyclin and isoproterenol. In a second system, a bioassay superfusion cascade procedure was employed to compare the relaxant effects and biologic stability of EDRF and NO. Acetylcholine and bradykinin elicited concentration-dependent but transient relaxant responses in arterial and venous rings, respectively. NO and S-nitroso-N-acetylpenicillamine, which generates NO in solution, elicited similar transient relaxant responses in endothelium-denuded arterial and venous rings. Glyceryl trinitrate, however, produced sustained relaxations, as did isoproterenol and prostacyclin. Utilizing a bioassay superfusion cascade system in which intact perfused artery or vein was the source of EDRF and three endothelium-denuded arterial or venous strips mounted in series served as the detector of EDRF or NO, the relaxation profile and biologic stability of superfused EDRF were compared with those of superfused NO. Arterial or venous perfusion with acetylcholine or bradykinin, respectively, and superfusion of NO over the strips produced characteristic decremental relaxant responses in the three vascular strips and revealed the highly unstable nature of EDRF and NO (T1/2 = 3-5 sec for both). The relaxation profile of EDRF was indistinguishable from that of NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular smooth muscle relaxation in response to chemically diverse naturally occurring neurotransmitters and autacoids has been attributed to the formation and/or release of one or more vascular endothelium-derived relaxing factors (EDRFs) distinct from prostacyclin. The chemical, biochemical, and pharmacological properties of one such EDRF resemble closely the properties of nitric oxide (NO). Thus, both arterial and venous EDRFs as well as authentic NO cause heme-dependent activation of soluble guanylate cyclase, endothelium-independent vascular and nonvascular smooth muscle relaxation accompanied by tissue cyclic GMP formation, and inhibition of platelet aggregation and adhesion to endothelial cell surfaces. EDRF from artery, vein, and freshly harvested and cultured aortic endothelial cells was recently identified as NO or a labile nitroso species as assessed by chemical assay and bioassay. Endothelium-derived NO (EDNO) has an ultrashort half-life of 3-5 s due to spontaneous oxidation to nitrite and nitrate, both of which have only weak biological activity. EDNO can be synthesized from L-arginine and possibly other basic amino acids and polypeptides, perhaps by oxidative metabolic pathways that could involve polyunsaturated fatty acid-derived oxygen radicals. Inorganic nitrite could serve as both a stored precursor and an inactivation product of EDNO. EDNO and related EDRFs may serve physiological and/or pathophysiological roles in the regulation of local blood flow and platelet function.

Recent studies have shown that N-nitroso compounds can activate arterial guanylate cyclase and relax isolated arterial smooth muscle; however, the effects of these substances on the cardiovascular system in the anesthetized cat are unknown. The present study was undertaken to compare the effects of several nitrosoguanidines and a nitrosamine, N-nitrosodimethylamine, on arterial guanylate cyclase activity, isolated arterial smooth muscle tone, and systemic vascular resistance in the anesthetized cat. Intravenous injections and infusions of the nitrosoguanidines glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) decreased systemic arterial pressure. During intravenous infusion of the nitrosoguanidines GTN and SNP, cardiac output was unchanged at the peak of the decrease in aortic pressure, indicating that the nitrosoguanidines GTN and SNP both reduced systemic vascular resistance. In addition, intraarterial injections of the nitrosoguanidines produced dose-dependent decreases in perfusion pressure in the feline mesenteric vascular bed perfused at constant flow. These substances were potent relaxants of isolated arterial smooth muscle and markedly activated arterial guanylate cyclase. In contrast, N-nitrosodimethylamine was devoid of vasodilator activity in vivo and exerted only minimal effects on isolated arterial smooth muscle tone or on arterial guanylate cyclase activity. The present data demonstrate a relationship between guanylate cyclase activation and arterial smooth muscle relaxation and suggest that the vasodilator effects on resistance vessels in vivo in response to selected N-nitroso compounds may involve such a mechanism. Although the significance of the presently reported cardiovascular responses to N-nitroso compounds is uncertain, N-nitroso compounds may represent a previously unrecognized class of substances which can be formed in the body and which possess marked vasodilator activity. It is possible that this vasodilator activity may involve the relaxation of vascular smooth muscle through activation of guanylate cyclase.

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 degrees C in a neutral, balanced salt solution containing glucose. Neutrophils ingested the particles and discharged beta-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 beta-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 beta-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.

A bioassay cascade superfusion procedure was utilized to study the release and effects of endothelium-derived relaxing factor (EDRF) from isolated bovine pulmonary artery and vein. Three precontracted, endothelium-denuded strips of pulmonary artery or vein mounted in series served as the detector of EDRF. Perfusion of artery with acetylcholine, bradykinin or A23187, in the presence of indomethacin, caused the release of EDRF (t1/2 = 3-5 s) which relaxed superfused strips of both artery and vein. Similarly, perfusion of vein with bradykinin or A23187, but not acetylcholine, caused the release of EDRF (t1/2 = 3-5 s) which relaxed superfused strips of both vein and artery. EDRF-elicited vascular smooth muscle relaxation was associated with cyclic GMP accumulation in the superfused strips. EDRF activity was increased and its duration prolonged by superoxide dismutase or low oxygen tension, whereas oxyhemoglobin and methylene blue were markedly inhibitory. The similar patterns of release, instability, and properties of EDRF from intact artery and vein suggest that arterial and venous EDRF are closely similar.

The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endothelium-denuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (t1/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.

The objective of this study was to elucidate the mechanisms by which bradykinin and vasoactive intestinal polypeptide (VIP) relax bovine intrapulmonary artery and bradykinin, but not VIP, relaxes intrapulmonary vein. Bradykinin and VIP elicited entirely endothelium-dependent relaxation of phenylephrine-precontracted arterial rings, and this was associated with arterial accumulation of both guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). Bradykinin, but not VIP, relaxed precontracted venous rings and increased cGMP, but not cAMP levels, by endothelium-dependent mechanisms. Neither arteries nor veins relaxed in response to substance P, thrombin, bombesin, arginine vasopressin, or angiotensin II. Methylene blue or indomethacin each partially antagonized, whereas both, when together, abolished arterial relaxant responses to bradykinin and VIP. Methylene blue or indomethacin, respectively, abolished arterial cGMP or cAMP accumulation elicited by bradykinin and VIP. Venous relaxation and cGMP accumulation elicited by bradykinin was abolished by methylene blue but was unaltered by indomethacin. Thus bradykinin and VIP relaxed bovine intrapulmonary artery by endothelium-dependent mechanisms involving the actions of cGMP and cAMP whose formation may be stimulated by endothelium-derived relaxing factor and prostacyclin, respectively. In contrast, bradykinin relaxed intrapulmonary vein by endothelium-dependent mechanisms involving only cGMP.

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containing forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 microM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 microM and 85-120 microM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 microM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg2+ or Mn2+. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO and nitroso compounds.

The influence of autonomic neurohormones on the immunologic release of beta-glucuronidase (EC 3.2.1.31) from, and the cyclic nucleotide levels in, human neutrophils was determined. Interaction of neutrophils with rheumatoid arthritic, serum-treated zymosan particles in a neutral balanced salt solution at 37 degrees resulted in the extracellular discharge of beta-glucuronidase without any loss of cell viability, as indicated by the failure of incubated cells to take up eosin Y or to release cytoplasmic lactate dehydrogenase (EC 1.1.1.27). Epinephrine reduced the release of beta-glucuronidase from neutrophils in the presence of zymosan during 2-30 min of incubation and elicited a concomitant elevation of adenosine 3':5'-monophosphate levels. Propranolol, a beta-adrenergic receptor antagonist, but not phentolamine, an alpha-adrenergic receptor antagonist, blocked both actions of epinephrine. Acetylcholine stimulated the release of beta-glucuronidase, but not lactate dehydrogenase, and provoked a concomitant elevation of guanosine 3':5'-monophosphate levels. Atropine, a muscarinic receptor antagonist, but not hexamethonium, a ganglionic blocker, inhibited both actions of acetylcholine. Interaction of neutrophils and zymosan particles resulted in an elevation of guanosine 3':5'-monophosphate levels within 2 min. These data suggest that intracellular guanosine 3':5'-monophosphate may be involved in mediating the immunologic release of lysosomal enzymes from human neutrophils whereas adenosine 3':5'-monophosphate may inhibit enzyme release. Moreover, autonomic neurohormones appear to be capable of modulating lysosomal enzyme release by virtue of their capacity to elevate neutrophil cyclic nucleotide levels.

The objective of this study was to ascertain whether "endothelium-derived relaxing factor" (EDRF) released from bovine intrapulmonary artery and vein is capable of directly activating soluble guanylate cyclase, thereby accounting for elevated vascular levels of cyclic GMP during EDRF release. Isolated arterial and venous rings, after equilibration and depolarization in bath chambers, were transferred to reaction tubes and incubated with soluble guanylate cyclase that had been purified to homogeneity from bovine lung. Addition of test agents to either bath chambers or enzyme reaction mixtures enabled the determination of their sites of action. Arterial and venous rings caused an endothelium-dependent 2- to 3-fold enzyme activation that was inhibited by methylene blue. Endothelium-dependent enzyme activation in artery but not vein was enhanced several-fold by acetylcholine in an atropine-sensitive manner. Bradykinin, which relaxes both artery and vein when endothelium is intact, activated guanylate cyclase upon addition of endothelium-intact rings to enzyme reaction mixtures. Vasoactive intestinal peptide, which causes endothelium-dependent relaxation of artery but not vein, also activated guanylate cyclase in the presence of endothelium-intact artery but not vein. Arachidonic acid activated the enzyme directly as well as through EDRF release from artery but not vein. Atrial peptides, prostacyclin, isoproterenol and nitroglycerin were inactive. Methylene blue was a powerful inhibitor of EDRF-elicited activation of guanylate cyclase but was without effect when rings were merely pretreated with methylene blue in bath chambers with no further addition to enzyme reaction mixtures. Thus, methylene blue did not interfere with the formation, release or chemical stability of EDRF, but rather inhibited its influence on guanylate cyclase. No agent was found to inhibit EDRF generation or release.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the endothelium in modulating cyclic nucleotide levels and intrinsic smooth muscle tone was studied in isolated rings of bovine intrapulmonary artery and vein. Cyclic 3',5'-guanosine monophosphate (cGMP) levels were threefold to fourfold higher in unrubbed artery and vein than in vessels that had been denuded of endothelium. Cyclic 3',5'-adenosine monophosphate (cAMP) levels were twofold higher in unrubbed than in endothelium-denuded artery, but no differences were observed in veins. Methylene blue, an inhibitor of guanylate cyclase, decreased cGMP but not cAMP levels, and this was accompanied by increases in smooth muscle tone. M&B 22,948, an inhibitor of cGMP-phosphodiesterase, increased cGMP but not cAMP levels, and this was accompanied by decreases in smooth muscle tone. Unrubbed vessels were more sensitive than endothelium-denuded vessels to the actions of both methylene blue and M&B 22,948, and this may be attributed to endothelium-dependent increases in cGMP turnover. Moreover, unrubbed vessels were more sensitive than endothelium-denuded vessels to contractile responses to phenylephrine and potassium, and these responses were potentiated by methylene blue and attenuated by M&B 22,948. Although indomethacin lowered cAMP levels in unrubbed artery, no changes in tone or contractile responsiveness were observed. A consistent observation was that the smaller branches of unrubbed but not endothelium-denuded intrapulmonary artery and vein had higher levels of cGMP but not cAMP, were sensitive to endothelium-dependent vasodilators, were more sensitive to methylene blue, and would not maintain a steady level of submaximal tone to phenylephrine when compared with larger branches from a common vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of the present study was to ascertain whether cyanide shares the properties of methylene blue as a selective inhibitor of vascular smooth muscle relaxation elicited by agents that stimulate the formation of cyclic GMP. Experiments were performed with endothelium-intact rings prepared from bovine intrapulmonary artery. Methylene blue, a good inhibitor of soluble guanylate cyclase, antagonized both arterial relaxation and cyclic GMP accumulation in response to sodium nitroprusside, glyceryl trinitrate, S-nitroso-N-acetylpenicillamine and acetylcholine. In contrast, cyanide inhibited only the responses to sodium nitroprusside. Increasing concentrations of methylene blue depressed resting arterial levels of cyclic GMP and caused slowly developing but marked contractions whereas cyanide was without effect. Contractile responses to phenylephrine, potassium and U46619 were potentiated by methylene blue but not by cyanide. Preincubation of dilute solutions of cyanide containing sodium nitroprusside in oxygenated Krebs' buffer at 37 degrees C for 15 min before addition to bath chambers depressed relaxation and cyclic GMP accumulation caused by sodium nitroprusside markedly. Similar treatment of glyceryl trinitrate, however, failed to alter its effects in arterial rings. A chemical inactivation of sodium nitroprusside by cyanide appears to account for the specific inhibitory action of cyanide on arterial responses to sodium nitroprusside. This study indicates clearly that cyanide does not share the properties of methylene blue as an inhibitor of arterial relaxation elicited by vasodilators that stimulate cyclic GMP formation.

The effects of acetylcholine chloride and isoproterenol on myocardiial cyclic GMP, cyclic AMP and on isometric tension were studied in isolated electrically driven rabbit atria. Acetylcholine (0.5 muM) produced a significant decrease in isometric force that was associated with a significant elevation in atrial cyclic GMP. Cyclic AMP was significantly lowered at 15 seconds after the addition of acetylcholine, but was only slightly decreased at earlier time periods. Both the negative inotropic action and increase in cyclic GMP after addition of acetylcholine were blocked by atropine. Isoproterenol (0.1 muM) produced a significant increase in isometric tension that was associated with a significant elevation in atrial cyclic AMP levels, whereas cyclic GMP levels were not changed. These effects were blocked by practolol. The increases in atrial cyclic GMP and cyclic AMP following addition of acetylcholine and isoproterenol, respectively, preceded the changes in isometric tension in response to these agents. These data support the hypothesis that changes in intracellular levels of cyclic AMP and cyclic GMP may mediate the positive and negative inotropic effects of adrenergic and cholinergic agents.

Mammalian tissues possess the capacity to synthesize cytidine 3',5'-monopHosphate (cyclic CMP) via the enzymatic conversion of cytidine 5'-triphosphate to cyclic CMP by cytidylate cyclase. Cyclic CMP formation occurs best in the presence of manganese or iron, at neutral pH, at 37 degrees C, in the absence of detergents, and with whole tissue homogenate fractions. Thus, mammalian tissues are capable of synthesizing not only cyclic AMP and cyclic GMP, but also cyclic CMP.

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 (35)S 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 (35)S 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 10(6) cells provoked two to four times more (35)S and beta-glucuronidase (beta-G) release from cartilage than phagocytic enzyme release conditions. beta-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 beta-G release by human cells in the presence of AHGG-coated cartilage was inhibited by 10(-5)M colchicine, whereas the protease activity, but not the release process, was inhibited by 10(-6)M 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.

Osmotic release of beta-glucuronidase from polymorphonuclear leukocyte lysosomes is inhibited by catecholamines and adenosine 3',5'-monophosphate, and accelerated by cholinergic agents and guanosine 3',5'-monophosphate. These actions are specific for the sympathetic and parasympathetic neurotransmitters and for the two cyclic nucleotides, as phenylephrine, tyramine, choline, adenosine 5'-monophosphate and guanosine 5'-monophosphate do not inodify lysosomal enzyme release.