Biochimica et Biophysica Acta 845 (1985) 223-236 223 Elsevier BBA 11478 Inhibition by verapamil of neutrophil responses to formylmethionylleucylphenylalanine and phorbol myristate acetate. Mechanisms involving Ca 2 + changes, cyclic AMP and protein kinase C Vittorina Della Bianca, Miroslawa Grzeskowiak, Pietro De Togni, Marco Cassatella and Filippo Rossi * Istituto di Patologia Generale, Unioersitit degli Studi di Verona, Strada 12 Grazie, 37134 Verona (Italy) (Received November 16th, 1984) Key words: Verapamil inhibition; fMet-Leu-Phe; Phorbol ester; Ca 2+ change; cyclic AMP; Protein kinase C; (Neutrophil) Verapamil inhibits in human neutrophils the respiratory burst, the secretion and the change of transmem- brane potential induced by formylmethionylleucylphenylalanine, a Ca2+-dependent stimulus, and by phorbol myristate acetate, a Ca2+-independent stimulus. Besides the blocking of Ca2+ channels, many mechanisms are responsible for the inhibition of neutrophil responses. In fact, verapamil (i) increases the intracellular cAMP concentration, potentiates the cAMP response induced by the chemotactic peptide and induces the appearance of a cAMP response also when the stimulant is phorbol myristate acetate; (ii) causes a decrease of Ca2+ association to cell membranes, so depleting the pools of exchangeable Ca2+ and depressing the 'Ca2+ response' in terms of rise in [Ca2+] i monitored with Quin 2 and of rapid mobilization from cell membranes monitored by chlorotetracycline fluorescence change; (iii) inhibits the Ca2+-activated phospholipid-dependent protein kinase C. The data, discussed in relation to the biochemical mechanisms of the stimulus-response coupling, are compatible with the hypothesis of an involvement of the activation of protein kinase C as key step in the sequence of transduction events for the induction of many neutrophil functions. Introduction The interaction of a variety of soluble and particulate agents with neutrophil plasma mem- brane induces cellular responses including chem- otaxis, endocytosis, secretion and respiration with production of oxygen radicals [1-5]. A number of biochemical events such as changes in Ca 2+ fluxes and mobilization from cellular stores [6-15], in- * To whom correspondence should be addressed. Abbreviations: fMet-Leu-Phe, formylmethionylleucylphenyl- alanine; PMA, phorbol myristate acetate; Hepes, N-2-hydroxy- ethylpiperazine-N-2-ethane-sulfonic acid; EGTA, ethylenegly- colbis(N, N'-tetraacetic acid). crease of phospholipid turnover [10, 15-22], activation of protein kinase C [23-26] and of protein phosphorylation [27-30] appear to be involved in the process of signal transmission for cell responses. In the course of investigations in our laboratory on the role of Ca 2+ as 'second messenger' for neutrophil functions, we have noted that the effect of some compounds did not correlate with their well-known activities. This was the case of verapamil, a widely known Ca 2+ channel blocker, [31-36], which in neutrophils influenced cellular responses induced also by CaE+-independent stimuli. On this basis, we investigated the effects of this drug on the biochemical mechanisms which 0167-4889/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)