Ronald G. Coffey

Stimulation of leukocyte adenyl cyclase by hydrocortisone and isoproterenol in asthmatic and nonasthmatic subjects

Abstract To evaluate the theory of beta-adrenergic blockade in asthma, we measured the effects of hydrocortisone and isoproterenol on adenyl cyclase in peripheral blood leukocytes from asthmatic and nonasthmatic children and nonasthmatio adults. An intact cell method was used, allowing cells to incorporate 3 H-adenine and convert it to 3 H-adenosine triphosphate (ATP) during a 2 hour incubation. Experimental compounds were then added for a pulse exposure of 5 minutes. AdenyZ cyclase catalyzes the transformation of 3 H-ATP to 3 H-cyclic adenosine monophosphate (AMP), which was measured after chromatographic separation. To rule out effects on rate of cyclic AMP destruction, aminophylline was routinely added at 25 mM., a concentration which completely inhibited cyclic AMP phosphodiesterase. Hydrocortisone at 10 −6 M stimulated adenyl cyclase in leukocytes from all subjects by 24 to 85 per cent. Isoproterenol at 10 −6 M stimulated adenyl cyclase by 16 to 19 per cent in control subjects, but not in cells from asthmatic subjects, a finding which supports the beta-adrenergic blockade hypothesis. Children receiving glucocorticosteroid therapy showed a restoration of leukocyte adenyl cyclase responsiveness to Isoproterenol. No synergistic effects between the steroid and catecholamine were observed in vitro. The data suggest at least 2 mechanisms by which gluoocorticosteroids benefit asthmatic patients: (1) Increased activity of adenyl cyclase, and (2) Restoration of adenyl cyclase responsiveness to catecholamines.

Endothelium-dependent vasorelaxation caused by various plant extracts

Summary: In a previous study (Am J Physiol 1993;265: H774–8), we found that certain red wines and other grape products caused endothelium-dependent vasorelaxation. In the present study, aqueous extracts of a variety of vegetables, fruits, teas, nuts, herbs, and spices were tested for their endothelium-dependent relaxing ability in vitro. Rings of rat aorta, with or without an intact endo-thelium, were mounted in tissue baths, contracted with phenylephrine, and then exposed to diluted plant extracts. Many, but not all, extracts exhibited endothelium-dependent relaxations that were reversed by NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, which suggested involvement of nitric oxide, the endo-thelium-derived relaxing factor in the response. Furthermore, extracts that caused relaxation also increased tissue levels of cyclic GMP, the mediator of nitric oxide-induced vascular smooth-muscle relaxation. These results may lend further support to mounting evidence that plant foods contain compounds that, if absorbed intact and in sufficient quantities, could conceivably be beneficial in prevention of cardiovascular disease.

Tetrahydrocannabinol inhibition of macrophage nitric oxide production.

delta 9-Tetrahydrocannabinol (THC) inhibited nitric oxide (NO) production by mouse peritoneal macrophages activated by bacterial endotoxin lipopolysaccharide (LPS) and interferon-gamma (IFN)-gamma). Inhibition of NO production was noted at THC concentrations as low as 0.5 microgram/mL, and was nearly total at 7 micrograms/mL. Inhibition was greatest if THC was added 1-4 hr before induction of nitric oxide synthase (NOS) by LPS and IFN-gamma, and declined with time after addition of the inducing agents. This suggested that an early step such as NOS gene transcription or NOS synthesis, rather than NOS activity, was affected by THC. Steady-state levels of mRNA for NOS were not affected by THC. In contrast, protein synthesis was inhibited as indicated by immunoblotting. NOS activity was also decreased in the cytosol of cells pretreated with THC. Addition of excess cofactors did not restore activity. Inhibition of NO production was greater at low levels of IFN-gamma, indicating the ability of the cytokine to overcome inhibition. The effectiveness of various THC analogues, in decreasing order of potency, was delta 8-THC > delta 9-THC > cannabidiol > or = 11-OH-THC > cannabinol. The presumably inactive stereoisomer, (+)delta 9-THC, and the endogenous ligand for cannabinoid receptors, anandamide, were weakly inhibitory. Inhibition may be mediated by a process that depends partly on stereoselective receptors and partly on a nonselective process. LPS, IFN-gamma, hormone receptor agonists, and forskolin increased macrophage cyclic AMP levels. THC inhibited this increase, indicating functional cannabinoid receptors. Addition of 8-bromocyclic AMP increased NO 2-fold, and partially restored NO production that had been inhibited by THC. This occurred only under conditions of limited NOS induction, suggesting that the effect of THC on cyclic AMP was responsible for only a small portion of the inhibition of NO.

The effect of phentolamine on adenylate cyclase and on isoproterenol stimulation in leukocytes from asthmatic and nonasthmatic subjects

Abstract Phentolamine, an alpha adrenergic antagonist, stimulated adenylate cyclase in human leukocytes, as determined by an increase in labeled cyclic adenosine monophosphate (CAMP) formed in intact cells that had incorporated 3 H-adenine. The response to phentolamine in leukocytes of asthmatic children was less than that in leukocytes of nonasthmatic children and adults. Phentolamine (2· × 10 −4 M) caused a mean increase in 3 H-cyclic AMP of 11.4, 30.7, and 42.7 per cent in leukocytes of asthmatic children, nonasthmatic children, and nonasthmatic adults, respectively. Propranolol blocked the increase of 3 H-cyclic AMP caused by phentolamine. The combination of phentolamine plus isoproterenol had an additive effect on 3 H-cyclic AMP formation in leukocytes of nonasthmatic children. The combination of phentolamine plus isoproterenol with leukocytes of asthmatic children produced a synergistic effect. Apparently phentolamine permitted an enhanced stimulation of adenylate cyclase by isoproterenol in leukocytes of asthmatics so that the isoproterenol effect reached the range seen with nonasthmatics. These results indicate that phentolamine can act as a beta adrenergic agonist and that it also facilitates isoproterenol responsiveness in leukocytes of asthmatic subjects, possibly by blockade of hypersensitive alpha adrenergic receptors.

Inhibition of macrophage nitric oxide production by tetrahydrocannabinol in vivo and in vitro

delta 9-Tetrahydrocannabinol (THC, 10 micrograms) was administered intraperitoneally to thioglycollate-treated mice. After 18 h, peritoneal macrophages were harvested and nitric oxide (NO.) production was induced by lipopolysaccharide (LPS, 1 microgram/ml) and interferon-gamma (IFN-gamma, 0.1-10 U/ml). Macrophages from THC-treated mice produced about half as much NO. as controls. THC (1 microgram/ml) added in vitro caused further inhibition. Greater inhibition was observed at the lower (0.1-0.3 U/ml) IFN-gamma concentrations. The results suggest that the use of THC can reduce NO. production and thereby affect host defense mechanisms, inflammation and autoimmune responses.

Is there a role for cyclosporine in asthma

Airway inflammation is a characteristic feature of asthma. After contact with a specific allergen on the surface of bronchial airway mast cells, IgE-dependent degranulation induces the early phase bronchial response, consisting of smooth muscle contraction and increased vascular permeability. The inflammatory LPR that begins 4 to 8 hours after the early phase is characterized by inflammatory cellular infiltrates in the bronchial mucosa and submucosa. The cells present in the bronchial airway wall and lumen, including mast cells, basophils, eosinophils, neutrophils, platelets, Tand B-lymphocytes, macrophages, and epithelial cells, are all involved in the pathogenesis of the inflammation in asthma. Although none of the cells can be identified as the primary effector cell, lymphocytes appear to have a critical role because of their immunoregulatory functions. Systemic glucocorticoids remain the cornerstone of the treatment of severe asthma in patients refractory to bronchodilator therapy. Their long-term use systemically, however, is associated with substantial and often irreversible adverse side effects. The favorable experience with nonsteroidal anti-inflammatory therapy of dermatologic and rheumatologic diseases has prompted studies of drugs in the treatment of severe asthma. Anti-inflammatory agents studied for their steroid-sparing capability include methotrexate, gold, nedocromil , colchicine, dapsone, hydroxychloroquine, and azathioprine, among other drugs. Only methotrexate, gold, and nedocromil have proven to

Increased Adenosine Triphosphatase in Leukocytes of Asthmatic Children

Adenosine triphosphatase (ATPase) activities were compared in leukocytes of asthmatic and nonasthmatic children. Both Mg(2+)- and Ca(2+)-dependent ATPase activities were significantly elevated in two membrane fractions (59 to 66%) and in a superntant fraction (68 to 72%) prepared from sonicated leukocytes of asthmatic subjects. Intact cell surface or ecto ATPase was also elevated (67 to 76%) in asthmatic leukocytes. Alternate day glucocorticosteroid therapy was associated with leukocyte ATPase activities intermediate between those for asthmatics not receiving steroids and for nonasthmatic control subjects. Incubation of normal leukocytes with 10(-8) M hydrocortisone or leukocyte membranes with 10(-4)-10(-3) M hydrocortisone in vitro also resulted in decreased ATPase activities. The elevated leukocyte ATPase activities appear to relate to the adrenergic imbalance in asthma previously characterized by reduced beta adrenergic responsiveness of adenylate cyclase and suggest the possibility of more than one enzymatic abnormality intrinsic to the asthmatic condition.

Release of Histamine from Rat Mast Cells by Lysosomal Cationic Proteins

Lysosomal cationic protein-mediated release of histamine from isolated rat peritoneal mast cells was inhibited by several compounds capable of increasing cellular levels of cyclic AMP. Histamine release was inhibited to a variable degree by 1–20 µm glucocorticosteroids, 0.05–1.0 mm catecholamines, 0.2–5.0 mm methylxanthines, and by 0.1–2.0 mm dibutyryl cyclic AMP. Β-adrenergic stimulation of adenylate cyclase by isoproterenol and Α-adrenergic stimulation of adenosine triphosphatase by norepinephrine were demonstrated in purified rat mast cells. These findings suggest that the regulation of histamine release by catecholamines may be mediated by Α- as well as by Β-adrenergic receptors. The ability of glucocorticosteroids to stimulate adenylate cyclase and to inhibit adenosine triphosphatase in mast cells suggests that steroids may influence histamine release by mechanisms similar to those involving Β-adrenergic agonists.

Methotrexate in bronchial asthma

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Prostaglandin-dependent desensitization of human monocyte cAMP responses.

Histamine stimulated large increases of cyclic adenosine monophosphate (cAMP) in freshly isolated human blood monocytes in the presence of RO2‐1724, a specific cAMP phosphodiesterase inhibitor. This was mediated by H2 receptors, since it was inhibited by cimetidine but not chlorpheniramine. Stimulation was attenuated in cells aged in culture 1–2 days. Indomethacin prevented the desensitization, suggesting that a cyclooxygenase product was responsible. Desensitization was heterologous, since the adenylate cyclase responses to 5′‐(N‐ethylcarboxamido)adenosine (A2 receptor agonist), isoproterenol (β‐adrenoceptor agonist), and prostaglandin E2 (PGE2) also declined during culture. The loss of sensitivity to histamine was restored by incubating monocytes with PGE2 in the presence of indomethacin. The results indicate that, while PGE2 inhibits monocyte functions via cAMP, its accumulation paradoxically permits cells to escape this regulation through a heterologous desensitization of the cAMP response to itself and other agonists.