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Effect of quercetin on human polymorphonuclear leukocyte lysosomal enzyme release and phospholipid metabolism
117 TEE EFFECT OF FLAVONOIDS ON CHEMILUMINESCENCE
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EFFECT OF ADRENERGIC AGENTS ON 45Ca UPTAKE BY RAT PERITONEAL MAST CELLS. K. Akagi, M.D., Y. Tanizaki~ M.D.~ R.G. Townley, M.D. Omaha, NE The purpose of the study was to determine if rat peritoneal mast cells were responsive to adrenergic agents. Mast cells were purified by bovine serum albumin gradients and stimulated with Con-A (i0 ug/ml) either in the presence or absence ~ adren~rgic agents. Isoproterenol (Iso, i0 - - i0- M) showed little effect except at high concentration (75.5 • 2.2% inhibition, mean + SE, i0- M). A similar effect was observed with d-lso. Thus the inhibitory effect of high concentration of Iso is presumed to be nonspecific. Norepinephrine (NE) exerted a dosedependent inhibitory effect at the concentrations from I0- -M to 10--M. This inhibition by NE ~as enhanced ~ith propranolol of NE except at i0- M. Prop (I0- M) potentiated ~ e effect of NE mainly at low concentrations I0-~- = IO-~M). Prazosin (Praz, 10- M) elicited little modulating effec~ on NE. On the other hand, yohimbine (Yoh, I0- M) attenuated the inhibitory effect of NE mainly at high concentration (i0 -~ - i0- M) of NE. (p < 0.05) % Inhibition of 45Ca Uptake Drug NE Conc. (M) Treatment n 10-12 I0Z8 10-4 Control 4 11.0• 33.1• 34.3• 51.4• Prop(10-~M) 4 34.5• 44.4• Prop(10-~M) 3 21.7• 34.1• 33.0• Praz(lO[JM) 4 14.8• 39.4• 40.0• Yoh(10 JM) 4 9.3• 22.6• 17.3• These results suggest that alpha adrenergic effects, especially alpha 2 type, are dominant on rat mast cells and may play an important role in the regulation of Ca channels.
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REPEATED SKIN TESTING IN GUINEA PIGS WITH FLUORESCEIN VISUALIZATION. J.H. Wells~ M.D.~ M. Lerner~ R. Strecker~ M.S.~ and W.A. Cain~ Ph.D., Oklahoma City, Oklahoma. During our chronic studies of immediate hypersensitivity in guinea pigs, a skin test procedure which could be repeatedly applied to the same animal became desirable. Sensitization is by weekly inhalation of aerosolized antigen extracts. Evidence of sensitization can often be found within 2 weeks after initial exposure. Methods for demonstrating sensitization have included direct and PCA skin testing, non-lnvasive serial (e.g. weekly) plethysmography during inhalation of antigen extracts, and antigen-induced histamine release from chopped lung tissue. Of these, direct skin titration is the most sensitive method for revealing and semi-quantitating early sensitization. When Evan's blue dye is used for either direct or PCA skin testing, the tested animal remains blue and is unusable for subsequent skin testing. Intracardiac fluorescein given immediately after antigen injection was found to delineate positive immediate hypersensitivity skin tests in ultra-violet light in a very satim factory fashion and can be used repeatedly in chronic experiments. With both direct and PCA skin tests the sensitivity is statistically similar to tests using Evan's blue dye. Fluorescein is rapidly eliminated and can be re-administered for additional skin testing by the following day. Methodology will be discussed further and photographic illustration of direct and PCA skin testing with antigen and histamine will be presented, comparing fluoresceln and Evan's blue dye.
(CL) GENERATION AND LYSOSOMAL ENZYME RELEASE BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES (PMN). D. EK o~p~ E. Middleton, Jr., M.D. and W.W. Busse, M.__~D., Madison, WI and Buffalo, NY Quercetin (Qu) is one of many naturally occurring flavonoid compounds that is a potent inhibitor of antigen-induced histamine release from human basophils (J IM~unol 127:546, 1981). Whether flavonoids have similar anti-inflammatory effects on other leukocyte functions is not established. The following study examined flavanoid modulation of granulocyte CL generation and lysosomal enzyme release. Human PMNs were isolated by Ficoll-Hypaque separation techniques. Luminol-dependent CL was measured during the incubation (37~ of PMNs with opsonized zymosan (Zm) particles. PMN membrane perturbation will stimulate the "respiratory burst" and with this is associated a light emission. A major component of CL involves superoxide generation through activation of the membrane NADPH-oxidase. Qu reduced CL in a dose-dependent fashion with maximal effects at 10"5M (72.4+8.3% inhibition). Similarly, inhibition occurred with the 8 other flavanoids tested. Zm particles also stimulate PMN lysosomal beta~glucuronidase (BG) release. Though Qu inhibited BG release, maximal reduction (34,2+5.4%) necessitated a 200 ~M dose. Interestingly, two flavanoids, hesperetin and catechin, consistently enhanced BG release. Therefore, two inflammatory PMN responses are inhibited by the majority of flavonoid compounds tested. Our findings also suggest that the PMN metabolic responses will vary in their sensitivity to the effects of individual flavanoid compounds.
11B EFFECTOF QUERCETINON HUMANPOLYMORPHONUCLEAR LEUKOCYTE LYSOSOMALENZYMERELEASEAND PHOSPHOLIPID METABOLISM. T.P. Lee~ M.L. Matteliano, and E. Middleton, Jr., Buffalo, NY. Flavonoids are naturally occurring chemicals widely distributed in the plant kingdom. Certain flavonoids, eg. quercetin, have been shown to affect various cellular functions including polymorphonuclear leukocyte (PMN) activation. To elucidate the biochemical mechanisms associated with the inhibitory action of quercetin on PMN function, we studied the effects of quercetin on PMN B-glucuronidase and arachidonic acid (AA) release. Humanperipheral blood PMNswere isolated by Ficoll-Hypaque separation and were suspended in Dulbecco's phosphate buffered saline (PBS) containing radioacive AA. After incubation (370 C, 60 min.) the cells were washed, resuspended in fresh PBS, and treated with different concentrations of quercetin followed by addition of zymosan-serum to i n i t i a t e B-glucuronidase and AA release. The purity of released AA and d i s t r i bution of radioactivity in different phospholipid fractions of cell l i p i d extractsv~re determined by thin layer chromatography. Release of AA accompanied release of B-glucuronidase and was associated with decreased radioactivity in several phospholipids. Quercetin inhibited the release of B-glucuronidase and AA and also i n h i b i ted the loss of radioactivity in phospholipids. As AA is preferentially incorporated into the Bposition of phospholipids, the results suggest that activation of PMNs is associated with activation of phospholipase A2 and release of AA and that the inhibitory effect of quercetin on Bglucuronidase release may be due to inhibition of phospholipase A2.
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119
EFFECT OF ADRENERGIC AGENTS ON 45Ca UPTAKE BY RAT PERITONEAL MAST CELLS. K. Akagi, M.D., Y. Tanizaki~ M.D.~ R.G. Townley, M.D. Omaha, NE The purpose of the study was to determine if rat peritoneal mast cells were responsive to adrenergic agents. Mast cells were purified by bovine serum albumin gradients and stimulated with Con-A (i0 ug/ml) either in the presence or absence ~ adren~rgic agents. Isoproterenol (Iso, i0 - - i0- M) showed little effect except at high concentration (75.5 • 2.2% inhibition, mean + SE, i0- M). A similar effect was observed with d-lso. Thus the inhibitory effect of high concentration of Iso is presumed to be nonspecific. Norepinephrine (NE) exerted a dosedependent inhibitory effect at the concentrations from I0- -M to 10--M. This inhibition by NE ~as enhanced ~ith propranolol of NE except at i0- M. Prop (I0- M) potentiated ~ e effect of NE mainly at low concentrations I0-~- = IO-~M). Prazosin (Praz, 10- M) elicited little modulating effec~ on NE. On the other hand, yohimbine (Yoh, I0- M) attenuated the inhibitory effect of NE mainly at high concentration (i0 -~ - i0- M) of NE. (p < 0.05) % Inhibition of 45Ca Uptake Drug NE Conc. (M) Treatment n 10-12 I0Z8 10-4 Control 4 11.0• 33.1• 34.3• 51.4• Prop(10-~M) 4 34.5• 44.4• Prop(10-~M) 3 21.7• 34.1• 33.0• Praz(lO[JM) 4 14.8• 39.4• 40.0• Yoh(10 JM) 4 9.3• 22.6• 17.3• These results suggest that alpha adrenergic effects, especially alpha 2 type, are dominant on rat mast cells and may play an important role in the regulation of Ca channels.
120
REPEATED SKIN TESTING IN GUINEA PIGS WITH FLUORESCEIN VISUALIZATION. J.H. Wells~ M.D.~ M. Lerner~ R. Strecker~ M.S.~ and W.A. Cain~ Ph.D., Oklahoma City, Oklahoma. During our chronic studies of immediate hypersensitivity in guinea pigs, a skin test procedure which could be repeatedly applied to the same animal became desirable. Sensitization is by weekly inhalation of aerosolized antigen extracts. Evidence of sensitization can often be found within 2 weeks after initial exposure. Methods for demonstrating sensitization have included direct and PCA skin testing, non-lnvasive serial (e.g. weekly) plethysmography during inhalation of antigen extracts, and antigen-induced histamine release from chopped lung tissue. Of these, direct skin titration is the most sensitive method for revealing and semi-quantitating early sensitization. When Evan's blue dye is used for either direct or PCA skin testing, the tested animal remains blue and is unusable for subsequent skin testing. Intracardiac fluorescein given immediately after antigen injection was found to delineate positive immediate hypersensitivity skin tests in ultra-violet light in a very satim factory fashion and can be used repeatedly in chronic experiments. With both direct and PCA skin tests the sensitivity is statistically similar to tests using Evan's blue dye. Fluorescein is rapidly eliminated and can be re-administered for additional skin testing by the following day. Methodology will be discussed further and photographic illustration of direct and PCA skin testing with antigen and histamine will be presented, comparing fluoresceln and Evan's blue dye.
(CL) GENERATION AND LYSOSOMAL ENZYME RELEASE BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES (PMN). D. EK o~p~ E. Middleton, Jr., M.D. and W.W. Busse, M.__~D., Madison, WI and Buffalo, NY Quercetin (Qu) is one of many naturally occurring flavonoid compounds that is a potent inhibitor of antigen-induced histamine release from human basophils (J IM~unol 127:546, 1981). Whether flavonoids have similar anti-inflammatory effects on other leukocyte functions is not established. The following study examined flavanoid modulation of granulocyte CL generation and lysosomal enzyme release. Human PMNs were isolated by Ficoll-Hypaque separation techniques. Luminol-dependent CL was measured during the incubation (37~ of PMNs with opsonized zymosan (Zm) particles. PMN membrane perturbation will stimulate the "respiratory burst" and with this is associated a light emission. A major component of CL involves superoxide generation through activation of the membrane NADPH-oxidase. Qu reduced CL in a dose-dependent fashion with maximal effects at 10"5M (72.4+8.3% inhibition). Similarly, inhibition occurred with the 8 other flavanoids tested. Zm particles also stimulate PMN lysosomal beta~glucuronidase (BG) release. Though Qu inhibited BG release, maximal reduction (34,2+5.4%) necessitated a 200 ~M dose. Interestingly, two flavanoids, hesperetin and catechin, consistently enhanced BG release. Therefore, two inflammatory PMN responses are inhibited by the majority of flavonoid compounds tested. Our findings also suggest that the PMN metabolic responses will vary in their sensitivity to the effects of individual flavanoid compounds.
11B EFFECTOF QUERCETINON HUMANPOLYMORPHONUCLEAR LEUKOCYTE LYSOSOMALENZYMERELEASEAND PHOSPHOLIPID METABOLISM. T.P. Lee~ M.L. Matteliano, and E. Middleton, Jr., Buffalo, NY. Flavonoids are naturally occurring chemicals widely distributed in the plant kingdom. Certain flavonoids, eg. quercetin, have been shown to affect various cellular functions including polymorphonuclear leukocyte (PMN) activation. To elucidate the biochemical mechanisms associated with the inhibitory action of quercetin on PMN function, we studied the effects of quercetin on PMN B-glucuronidase and arachidonic acid (AA) release. Humanperipheral blood PMNswere isolated by Ficoll-Hypaque separation and were suspended in Dulbecco's phosphate buffered saline (PBS) containing radioacive AA. After incubation (370 C, 60 min.) the cells were washed, resuspended in fresh PBS, and treated with different concentrations of quercetin followed by addition of zymosan-serum to i n i t i a t e B-glucuronidase and AA release. The purity of released AA and d i s t r i bution of radioactivity in different phospholipid fractions of cell l i p i d extractsv~re determined by thin layer chromatography. Release of AA accompanied release of B-glucuronidase and was associated with decreased radioactivity in several phospholipids. Quercetin inhibited the release of B-glucuronidase and AA and also i n h i b i ted the loss of radioactivity in phospholipids. As AA is preferentially incorporated into the Bposition of phospholipids, the results suggest that activation of PMNs is associated with activation of phospholipase A2 and release of AA and that the inhibitory effect of quercetin on Bglucuronidase release may be due to inhibition of phospholipase A2.
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