Inhibition of myeloperoxidase release from rat polymorphonuclear leukocytes by a series of azachalcone derivatives.

A series of azachalcones was evaluated for ability to affect secretion of myeloperoxidase by rat polymorphonuclear leukocytes stimulated by fMLP. The compounds were found to interfere with cellular uptake of extracellular calcium. Structure-activity relationships are discussed.

The antinociceptive activity of paracetamol in zymosan-induced peritonitis in mice: the role of prostacyclin and reactive oxygen species.

1. Oral administration of high doses of paracetamol (600 mg kg-1 or more) resulted in inhibition of the writhing and reduced the levels of prostacyclin (PGI2, measured as 6-keto-PGF1 alpha) induced by intraperitoneal administration of zymosan in mice. The high oral doses of paracetamol required were accompanied by behavioural toxicity which may have contributed to the inhibition of writhing. 2. The number of writhes per mouse and the proportion of mice writhing at least once correlated significantly with the levels of 6-keto-PGF1 alpha. However, inhibition of writhing by paracetamol occurred at higher levels of 6-keto-PGF1 alpha than was previously observed with acidic non-steroidal anti-inflammatory agents. 3. When injected i.p., PGI2, carbacyclin and iloprost (agonists at the PGI2 receptor) induced writhing. Intraperitoneal injection of PGI2 reversed the inhibition of writhing induced by indomethacin (1 mg kg-1, p.o.) but not that induced by oral administration of paracetamol. 4. Paracetamol at 800 mg kg-1, p.o., inhibited carbacyclin-induced writhing but indomethacin at 1 mg kg-1 p.o. did not. Paracetamol administered i.p. at 100 mg kg-1 reduced the peritoneal levels of 6-keto-PGF1 alpha and inhibited zymosan-induced but not carbacyclin-induced writhing and did not produce behavioural toxicity. 5. The in vitro potency of paracetamol as a prostaglandin synthesis inhibitor is known to be reduced by the presence of lipid peroxides. However, no lipid peroxides, measured as thiobarbituric acid reactive material, were detected in the peritoneal lavage fluid of zymosan-injected mice. 6. Intraperitoneal administration of a mixture of superoxide dismutase and catalase reduced detectable superoxide anion by 98% without inhibiting the writhing response to zymosan or the antinociceptive potency of paracetamol. 7. The data are consistent with the suggestion that inhibition of PGI2 synthesis in the peritoneal cavity by paracetamol is responsible for only a part of its antinociceptive activity in this test. However, extremely high oral doses of paracetamol were required which produced behavioural toxicity which clearly contributed to the inhibition of writhing. The low potency of paracetamol in this model cannot be attributed to the generation of lipid peroxides via the oxidative burst.

Ex vivo lipopolysaccharide-induced interleukin-1 secretion from murine peritoneal macrophages inhibited by probucol, a hypocholesterolemic agent with antioxidant properties.

Probucol, 4,4'-(isopropylidenedithio)bis(2,6-di-tert-butyl-phenol), has been shown to inhibit atherogenesis in genetically hypercholesterolemic (Watanabe) rabbits. Since atherosclerotic lesions contain macrophages capable of screting interleukin 1 (IL 1) and other cytokines that could contribute to the pathogenesis of the disease, we have investigated whether probucol affects IL 1 secretion. Resident peritoneal macrophages from mice dosed with probucol secreted 40-80% less IL 1 than macrophages from control animals when stimulated in vitro with lipopolysaccharide (LPS). The inhibitory effect of probucol was observed when IL 1 was assayed by the standard bioassay, the thymocyte proliferation assay, or a competitive IL 1 receptor binding assay. Probucol treatment had no effect on LPS-induced membrane IL 1 expression; secretion of tumor necrosis factor (TNF); Con A-induced splenic interleukin 2 (IL 2) and interleukin 3 (IL 3) release; and prostaglandin- or zymosan-induced secretion of prostacyclin, leukotriene C4, acid phosphatase, or superoxide anion. In contrast to the effect of oral administration, direct addition of probucol to macrophage cultures did not inhibit IL 1 release. Probucol administration did, however, inhibit the fall in serum zinc level induced by intravenous injection of LPS in zymosan-primed mice but had no effect on the LPS-induced increase in serum triglyceride levels, which indirectly confirms that probucol administration inhibits IL 1 but not TNF secretion. Paw granuloma induced in mice by heat-killed mycobacteria was inhibited by oral administration of probucol, an effect that may be attributable to inhibition of IL 1 secretion. Probucol neither reduced zymosan-induced liver granulomata in mice nor inhibited adjuvant-induced arthritis in rats. We suggest that inhibition of IL 1 secretion from macrophages by probucol contributes to its therapeutic effects in atherosclerosis and may also result in beneficial activity in some chronic inflammatory diseases.

Novel inhibitors of polymorphonuclear neutrophil (PMN) elastase and cathepsin G: evaluation in vitro of their potential for the treatment of inflammatory connective tissue damage.

Inhibitors of neutrophil proteases may have therapeutic effects in inflammatory diseases. MDL 27,324 (Dansyl-Ala-Ala-Pro-Val-CF3), inhibits human neutrophil elastase and MDL 27,399 (MeOSucc-Ala-Ala-Pro-Phe-COOCH3), inhibits human neutrophil cathepsin G. These compounds individually or in combination, partially inhibited the hydrolysis of fluoresceinated bovine serum albumin and fluoresceinated immune complexes by rat and human neutrophil granule lysate. In contrast, the combination of inhibitors completely prevented the breakdown of a complex connective tissue substrate, azure hide powder. Rat neutrophils phagocytosed and hydrolyzed fluoresceinated immune complexes, a process which was inhibited by cytochalasin B (15 micrograms/ml, 65% inhibition) and chloroquine (200 microM, 80% inhibition). Although MDL 27,324 was taken up by the cells, it had only a modest inhibitory effect on the proteolysis of ingested fluoresceinated immune complexes (200 microM, 20% inhibition); MDL 27,399 had similar limited efficacy. Therefore, these compounds may be effective inhibitors of neutrophil serine proteases secreted into the extracellular space during inflammation without interfering with the normal process of intracellular degradation of phagocytosed material.

Exogenous l-dopa alters spiroperidol binding, in vivo, in the mouse striatum.

The effect of exogenous l-dopa on the binding of tritiated spiroperidol, in vivo, in the murine striatum was investigated. The results obtained demonstrated that the binding of 3H-spiroperidol can be altered by the administration of l-dopa. This effect appears to be related to both the dose of spiroperidol and l-dopa given. Since l-dopa raised striatal dopamine levels in a dose dependent manner, these results suggest that the estimates of receptor parameters obtained using positron emission tomography and analogs of spiroperidol may be affected by the differences in endogenous dopamine concentrations among subjects.

Intracellular pH in human fibroblasts: effect of mitogens, A23187, and phospholipase activation.

Serum-deprived human fibroblasts (HSWP cells) were loaded with either the fluorescent pH indicator 6-carboxy-4',5'-dimethylfluorescein or the calcium indicator quin 2, and the fluorescence of the intracellular probes was continuously monitored with a microspectrofluorometer. Addition of a cocktail of peptide growth factors causes intracellular alkalinization, which is blocked by amiloride or by replacement of extracellular Na+ with choline, confirming that mitogenic stimulation activates a Na+-H+ exchanger in HSWP cells. The exchanger is also activated by A23187, acid loading the cells, and stimulation of phospholipase activity with melittin. Growth factors and melittin activation of the exchanger have been demonstrated to be dependent on the mobilization of Ca2+ from intracellular stores. The intracellular alkalization and increase in Ca2+ activity due to both melittin and growth factors is inhibited by phospholipase inhibitors, indicating that phospholipase activity is necessary for the activation of the Na+-H+ exchanger and the mobilization of intracellular Ca2+.

In vivo binding of spiroperidol and bromospiroperidol at low drug loadings.

The binding of spiroperidol and bromospiroperidol, in vivo, was studied over a wide range of drug dosages. It was found that while spiroperidol and bromospiroperidol bind selectively in vivo to tissues known to be high in dopamine receptor binding sites, this specificity of binding does not persist at very low doses. Such anomalous binding behavior can have implications for the non-invasive imaging of these drugs.

Depolarization-induced increases in intracellular free calcium detected in single cultured neuronal cells.

Microspectrofluorometry was used to examine intracellular free calcium changes in single NG108-15 neurons loaded with the Ca2+ sensitive probe, quin2. The changes in intracellular free Ca2+ were induced either by depolarizing cells with high K+ or veratridine or by the addition of ionomycin. Ca2+-free medium and various inorganic and organic calcium-channel blocking agents blocked changes in intracellular free Ca2+ levels, indicating that Ca2+ entry is most likely through voltage-sensitive calcium channels.

Morphologic and cytochemical characteristics of amine-containing globule leukocytes in rat tracheal epithelium.

Amine-containing cells in the tracheal epithelium are typically of the small-granule type (diameter approximately 100 nm). However, in the rat, another amine-containing cell type has been identified that possesses the amine-handling features of the APUD-series of cells (amine precursor uptake and decarboxylation) but not the ultrastructural characteristics. It has been postulated that these cells may be related to cutaneous melanocytes. In this study, fluorescent cells were present in the laryngeal and tracheal epithelial lining of adult Sprague-Dawley rats following freeze-drying and exposure to formaldehyde vapor (FIF or formaldehyde-induced fluorescence). Microspectrofluorimetry revealed an emission maximum at 493 nm. The excitation maximum could not be calculated but appeared to be around or below 350 nm (to record spectra below requires the use of quartz optics). Yellow fluorescence also emanated from serotonin-containing mast cells (excitation and emission maxima: 401/515 nm). Tracheal segments processed according to the aqueous formaldehyde ( AFIF ) technique, for the demonstration of 5- hydroxytryptophan (5-HTP) or serotonin (5-HT), failed to identify fluorescent cells in the epithelial lining even though connective-tissue mast cells were evident. Subsequent treatment of AFIF -fixed sections with formaldehyde and HCl vapors ( AFIF -HCl) resulted in the formation of a fluorogenic compound within numerous cells in the tracheal lining (455/537 nm). This spectral shift and increase in intensity of fluorescence following acidification are characteristic for standards and/or cells that contain tryptamine, tryptophan, or peptides with NH2-terminal tryptophan and are markedly different from microspectrofluorimetric data reported for the phenylethylamines or serotonin. It is therefore postulated that these cells contain a closely related beta-(3-indolyl) ethylamine-like compound, serotonin excluded. The morphology of the fluorescent cells was similar when prepared according to the FIF or AFIF -HCl techniques. Conjunctive staining, the examination of a single section first by fluorescence microscopy and subsequently by other histochemical and cytochemical methods, demonstrated that the fluorescent granules were also methylene blue, alcian blue, periodic-acid Schiff, and ferric- fericyanide positive. Subsequent correlative electron microscopic examination of Epon-embedded AFIF -HCl-treated tracheal sections demonstrated that these amine-containing cells were globule leukocytes.

Mitogens and melittin stimulate an increase in intracellular free calcium concentration in human fibroblasts.

Intracellular free Ca+2 concentration was measured in cultured human fibroblasts (HSWP) utilizing the Ca+2-sensitive fluorescent probe quin2. The addition of peptide growth factors to serum-deprived HSWP cells induced an immediate rise in intracellular Ca+2 concentration. This mitogen-induced rise in Ca+2 concentration could be blocked by the addition of the intracellular Ca+2 antagonist TMB-8. Addition of the phospholipase activator, melittin, to cells in the absence of growth factors also caused a dramatic rise in intracellular Ca+2 concentration which was blocked by TMB-8.

Measurements in vivo of parameters of the dopamine system.

An in vivo study of the binding of the neuroleptic drugs spiroperidol and bromospiroperidol has indicated a large amount of specific binding to the dopamine-rich caudate nuclei of the murine brain. We have tested the applicability of the simple thermodynamic equilibrium equation to describe this binding and find that it seems to be a reasonable description of the process. It appears that a period of several hours is needed for reaching equilibrium, but after that time the behavior appears slowly to follow the equilibrium isotherm. One consequence of this model is the apparent competition of endogenous dopamine for the binding sites in vivo. The data can only be fitted by the calculations when the endogenous dopamine is included as a competitive ligand. This competition presents the interesting possibility of measuring the synaptic dopamine concentration by its effect on the binding of neuroleptic drugs. A second observation in these studies was the increase of nonspecific binding of bromospiroperidol at very low drug loadings. This increase is attributed to the presence of binding sites that are ubiquitously dispersed and very dilute but of high affinity.

Evidence for dopamine-containing renal nerves.

The existence of the specific renal DA1 postsynaptic receptor for dopamine (DA) has prompted the search for a counterpart dopaminergic innervation. In the canine kidney there is an increased proportion of DA as a percentage of norepinephrine (NE), and both NE and DA are lost after chronic denervation. In the rat and dog, renal stimulation increases the net secretion of both NE and DA; renal denervation eliminates NE but only partially reduces DA secretion. Histological techniques have identified DA-containing neuronal elements in the kidney. Thus there is growing evidence for a prejunctional dopaminergic counterpart to the DA1 receptor.

Site of estrogen action in rabbit ovarian tissue.

In superovulated or naturally mated pseudopregnant rabbits, an abrupt premature decline of progesterone production by the corpus luteum occurs after removal of polydimethylsiloxane capsules filled with 17 beta-estradiol. Reinsertion of the estradiol-filled capsules stimulates a striking restoration of progesterone production. We have given [3H]estradiol by arterial infusion to anesthetized rabbits to determine by cellular autoradiography whether the exogenous estradiol localizes to luteal tissues responsible for progesterone production. Autoradiography with both freeze-dried and touch-thaw mounted preparations demonstrated that tritium activity localized to the nuclei of luteal cells and that such uptake could be blocked by radioinert diethylstilbestrol. We extracted the tritium activity sequestered in vivo by the corpora lutea and cocrystallized the extract with 14C-labeled and radiostable 17 beta-estradiol to confirm its presence (greater than 90%) as [3H]estradiol. In addition, we were able to demonstrate that radiostable diethylstilbestrol blocked ovarian uptake of 16 alpha-125I-17 beta-estradiol in vivo. In concert, these observations provide compelling evidence that the exogenous estradiol responsible for restoring progesterone production may act through an estrogen-specific mechanism directly in the luteal cells.

Morphology, histochemistry, and distribution of serotonin-containing cells in tracheal epithelium of adult rabbit.

We have investigated morphologic and histochemical characteristics of serotonin-containing epithelial cells in tracheas from adult rabbits, using the Falck-Hillarp freeze-dried formaldehyde vapor technique. An intracellular formaldehyde-induced fluorescent substance was identified as serotonin by microspectrofluorometric techniques. Fluorescence microscopy and subsequent histochemical staining of the same sections demonstrated that serotonin-containing cells were argentaffin-, argyrophil-, and ferric ferricyanide-positive. The serotonin-containing epithelial cells were more numerous in ventral than in dorsal aspects of trachea. The number of detectable fluorescent cells was reduced after reserpine administration but was not affected by injecting the amine precursor L-dihydroxyphenylalanine (L-DOPA). The emission peak of the fluorophore was not significantly shifted after L-DOPA injections. The cells may regulate tracheobronchial-pulmonary function by releasing serotonin or other as yet unidentified biologically active substances.

Identification and localization of glucagon-related peptides in rat brain.

Immunochemical and immunocytochemical techniques have been used to identify and characterize glucagon-related peptides of the rat central nervous system. These peptides show immunoreactivity with antiglucagon sera directed towards the central portion of the hormone, but not with antisera specific for the free COOH terminus of glucagon. Highest concentrations were found in hypothalamus (6.1 +/- 1.6 ng/g wet weight) although lower amounts (approximately 2 ng/g) were found in cortex, thalamus, cerebellum, and brain stem. Gel filtration of brain extracts revealed at least two immunoreactive forms, which have molecular weights of about 12,000 and 8000. Both peptides had radioimmunoassay dilution curves parallel to the curve for glucagon and both had identical counterparts in extracts of rat intestine. Digestion of the brain and intestinal peptides with trypsin plus carboxypeptidase B released the immunoreactive COOH-terminal tryptic fragment of pancreatic glucagon from these larger forms. Immunocytochemical studies using antiglucagon serum and peroxidase-antiperoxidase staining identified glucagon-like material in neuronal cell bodie and processes in the magnocellular portion of the paraventricular nucleus, as well as in scattered cells in the supraoptic nucleus and in fibers in the median eminence. These results suggest that glucagon-containing peptides that have undergone the intestinal type of posttranslational modification are present in neuronal cells of the rat hypothalamus.

Histofluorescence techniques provide evidence for dopamine-containing neuronal elements in canine kidney.

Changes induced by hydrochloric acid in the excitation spectrum of catecholamine fluorophores associated with the innervation of the canine renal vasculature show that there are neuronal elements at the glomerular vascular poles containing predominantly dopamine. In contrast, the catecholamine fluorescence in the periadventitial layer of the arcuate arteries is derived from norepinephrine. The dopamine-containing structures may represent the prejunctional counterpart to the pharmacologically identified dopamine receptors in the renal vasculature. As such, this system may be involved in the normal regulation of renal blood flow and renin release.

An artifact of liquid emulsion autoradiography.

The localization of 3H-opiates in the myenteric plexus of the guinea pig ileum is subject to systematic artifact when stretch preparations of the myenteric plexus are dipped into liquid Kodak NTB-3 emulsion for autoradiography. The cause of the artifact was determined to be a discontinuous distribution, or retraction, of emulsion over plexuses. The apposition of frozen freeze-dried ileal sections to dried photographic emulsion avoids this source of error.