A comparison of contractile events induced by leukotriene D4 and histamine in smooth muscle—A function of calcium utilization

Abstract

OF CONTRACTILE EVENTS INDUCED BY LEUKOTRIENE D 4 AND HISTAMINE IN SMOOTH MUSCLE-A FUNCTION OF CALCIUM UTILIZATION. S. R. Findlay, C.W. Parker, E.I. Bloomquist~ and C.R. Scheid~ Tufts University Sch06l of'Medicine, Boston, MA. Washington University School of Medicine, St. Louis, MO. and the University of Massachusetts Medical School, Worcester, MA. Studies characterize the time course of exogenous LTD4-induced smooth muscle contraction in guinea pig i leal longitudinal smooth muscle. Tissues were prepared and secured in a 37oc constantly aerated (95% 02-5% CO2) tissue bath. Parameters characterizing the speed of contraction were: latency time-from the agonist addition to force development; T 89 half the time between the end of L and maximum force; relaxation time (Tr) ended when tissues reached baseline passive tension (0.5 gm) after washing. LTD4 as compared to histamine had a longer latency time, a markedly longer T 89 Tm and Tr. For LTD4 (1.4 x lO-9M) Jn sec. + S.D. L=6.3+I.6, T 89 4.1, Tm= 84+47, Tr= 136u and forhistamTne L = 4.5+I.8, T 89 1.8+0.8, Tm 4.2+0.9, Tr= 12+6. Compared to histamine, LTD 4 appeared to activate only a late tonic component of contraction. Histamine induced an early phasic and late tonic component of contraction. The ID50 of D600 (a calcium channel antagonists) for histamine and LTD 4 contractile components supported the concept that the slowness of the onset of contraction is largely due to the inabi l i ty of LTD 4 to mobilize Ca z+ via the mechanisms used by histamine in the production of the phasic component of contraction. METABOLISM OF HUMAN C3 BY HUMAN MAST CELL TRYPTASE. L.B. Schwartz, M.D., Ph.D., J.J. Schratz, D. Vik, D.T. Fearon, M.D. and K.F. Austen, M.D., Boston, Massachusetts Human mast cell tryptase was assessed for peptidase act iv i ty against human C3 and the synthetic C3 analog, leu-ala-arg-p-nitroanalide (LAA) (Kabi) and esterase act iv i ty against tosyl-L-arg methylester (TAMe) and CBZ-lys-thiobenzyl ester (CLTE). Tryptase was purified to homogeneity after extraction from dispersed and concentrated pulmonary mast cells of 10-35% purity by chromatography on Dowex I-x2, DEAE-Sephadex and heparin-agarose. One unit (U) of act iv i ty catalyzes the cleavage of 1 ~mole of substrate/min at 22~ Human C3 was purified as in Biochemistry 15:4513, 1976. Michaelis Menton kinetics were observed with all synthetic substrates. Ester substrates at pH 8.1 had respective Km and Vmax values for TAMe of 0.35 mM and 124 U/mg and for CLTE of 0.024 mM and 118 U/mg. LAA at pH 7.4 had a Km of 2 mM and Vmax of 104 U/mg. Cleavage of C3 was assessed by incubation of 2 ~g of tryptase and 126 ug of C3 in 0.42 ml of 0.01 M Tris, pH 7.8, 0.15 M NaCI, 2 mM CaCl 2 at 37~ aliquots removed at time points up to 12 h were analyzed by SDS-PAGE under reducing conditions. The 70,000 MW 8 chain was not cleaved. Several cleavage products of the 110,000 MW ~ chain were detected at 26,000-38,000 MW and a single product was observed at about 9000 MW. The lower MW product had the same apparent MW as trypsingenerated C3a. Thus, tryptase from human mast cell secretory granules can metabolize human C3 and generate protein fragments which may amplify mast cell init iated reactions by a histamineindependent mechanism.