Effect of Cystathionine Ketimine on the Stimulus Coupled Responses of Neutrophils and Their Modulation by Various Protein Kinase Inhibitors Jianying Zhang,* Kazunori Sugahara,* Yasuhiro Sagara,† Mario Fontana,‡ Silvestro Dupre `,* and Hiroyuki Kodama* ,1 *Departments of Chemistry and †Medical Biology, Kochi Medical School, Okocho, Nankoku, Kochi 783, Japan; ‡Dipartimento di Scienze Biochimiche, Università di Roma “La Sapienza,”, Piazzale Aldo Moro 5, 00185 Rome, Italy Received November 25, 1995 Human peripheral blood polymorphonuclear leukocytes were preincubated with cystathionine and cystathio- nine metabolites found in the urine of the patients with cystathioninuria. Among the cystathionine metabolites, cystathionine ketimine significantly enhanced the N-formyl-methionyl-leucyl-phenylalanine-induced superoxide generation, but cystathionine and cyclothionine did not enhance the superoxide generation. Cystathionine keti- mine also enhanced superoxide generation induced by opsonized zymosan but not those induced by arachidonic acid and phorbol myristate acetate. Superoxide generation induced by cystathionine ketimine was inhibited by genistein, an inhibitor of tyrosine kinase, and was enhanced by 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine, an inhibitor of protein kinase C. © 1995 Academic Press, Inc. Polymorphonuclear leukocytes (PMN) play critical roles in the defense mechanism against microorganisms [1]. When PMN are exposed to various stimuli, one-electron reduction of mo- lecular oxygen by NADPH-oxidase leading to “respiratory burst” is induced [2, 3]. N-formyl- methionyl-leucyl-phenylalanine (fMLP), opsonized zymosan (OZ), arachidonic acid (AA) and phorbol 12-myristate 13-acetate (PMA) are known as the stimuli [4]. Although the responsibility of PMN to agonist is low in the basal condition, preincubation of PMN with non-stimulatory concentrations of agonists or some pharmacological agents and hypotonic treatment of the cells accelerate and potentiate the respiratory burst induced by a second stimulus [5–12]. This phenom- enon, termed “priming”, may account for the exaggerated physiological responses of human peripheral blood polymorphonuclear leukocytes (HPPMN). Priming and stimulation of PMN have been proposed to occur through different mechanisms [13, 14]. Recently, several reports described that various cytokines and hypotonic condition enhanced the tyrosyl phosphorylation of specific proteins in PMN in the primed stage, suggesting the contribution of tyrosine kinase (TK) to the regulatory mechanism of priming in neutrophils [11, 14–16]. We found that the iminodipeptides such as Pro-Pro enhanced the superoxide (O 2 - ) gen- eration induced by fMLP or OZ but not that induced by AA or PMA and that tyrosyl phosphor- ylation of 45-kDa protein occurred in parallel with the iminodipeptide-dependent enhancement of O 2 - generation in neutrophils [17]. These results also suggest that TK may play critical role(s) in priming and activation of NADPH-oxidase. However, the precise mechanism and the role of tyrosyl phosphorylation in priming and activation of neutrophils are unknown yet. Cystathioninuria is an autosomal recessive hereditary disorder and the phenotypical homozy- gotes lead to persistent excretion of large amounts of cystathionine in the urine owing to cysta- 1 To whom correspondence should be addressed. Fax: +81-888-80-2281. Abbreviations: PMN, polymorphonuclear leukocytes; fMLP, N-formyl-methionyl-leucyl-phenylalanine; OZ, opsonized zymosan; AA, arachidonic acid; PMA, phorbol 12-myristate 13-acetate; HPPMN, human peripheral blood polymorpho- nuclear leukocytes; TK, tyrosine kinase; CK, cystathionine ketimine; H-7, 1-(5-isoquinoline-sulfonyl)3-methyl-piperazine; NAc-cysta, N-acetylcystathionine; CMHC, S-(carboxymethyl) homocysteine; HCEHC, S-(2-hydroxy-2-carboxy- ethyl)homocysteine; -CEC, S-(2-carboxyethyl)cysteine; HCPC, S(3-hydroxy-3-carboxy-n-propyl)cysteine; OCEHC, S-(2- oxo-2-carboxyethyl)homocysteine; CT, cyclothionine; PKC, Ca 2+ - and phospholipid-dependent protein kinase. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 218, 371–376 (1996) Article No. 0065 371 0006-291X/96 $12.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.