Initial stages of programmed cell death (apoptosis) possess tissue specific features that are lost at the terminal (universal) stages during activation of specific proteases (caspases). This is important for the development of pharmacological approaches for correction of diseases (e.g., Alzheimer’s and Parkinson’s diseases) character- ized by premature cell death or accumulation of “unwanted” cells that appear during malignant conver- sion or tissue hypertrophy. Long-term maintenance of increased blood pressure and atherosclerosis are accom- panied by vascular wall hypertrophy [1-4]. We earlier investigated the role of signal systems in the development of apoptosis of vascular smooth muscle cells (VSMC). We found that in contrast to immune system cells, VSMC are less sensitive to agonists of so-called death receptors (FAS-ligand, TNF-α); however, these cells undergo apoptosis in growth factor-free medium. Addition of the low-selectivity protein kinase inhibitor staurosporine and the highly selective inhibitors of serine/threonine phos- phatases caliculin and okadaic acid also induced apopto- sis of VSMC [5]. Activation of the cAMP signaling system [6] or increase of intracellular [Na + ]/[K + ] ratio [7] atten- uated the development of apoptosis of VSMC at the stage preceding caspase-3 activation. However, systems involved in protection of cells against apoptosis are tissue- specific. For example, Na + /K + -ATPase inhibitors exhibit the antiapoptotic effect in VSMC and neuronal cell cul- ture [8], but they were ineffective in immune system cells pretreated with FAS-ligand [7]. Antiapoptotic effect of activators of cAMP signaling was noted in neutrophils [9], hepatocytes [10], and neuronal [11] and endothelial [12] cells but not in T-lymphocytes [13]; moreover, in thymocytes activation of cAMP signaling stimulated apoptosis [14]. Biochemistry (Moscow), Vol. 67, No. 2, 2002, pp. 254-259. Translated from Biokhimiya, Vol. 67, No. 2, 2002, pp. 303-310. Original Russian Text Copyright © 2002 by Taurin, Ryazhsky, Maximova, Chuchalin, Hamet, Pshezhetsky, Orlov. 0006-2979/02/6702-0254$27.00 ©2002 MAIK “Nauka / Interperiodica” * To whom correspondence should be addressed. Suppression of Programmed Cell Death by Intracellular cAMP Is not Mediated by Expression of Genes Encoding an Inhibitor of Apoptosis S. Taurin 1 , G. G. Ryazhsky 2 , N. V. Maximova 2 *, A. G. Chuchalin 1 , P. Hamet 1 , A. V. Pshezhetsky 1 , and S. N. Orlov 1,2 * 1 University of Montreal Research Center, Quebec, Canada 2 Laboratory of Physical Chemistry of Biomembranes, School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-1116; E-mail: sergei.n.orlov@umontreal.ca Received November 22, 2000 Revision received May 17, 2001 Abstract—The elevation of intracellular cAMP content is accompanied by expression of genes whose promoter contains a Ca 2+ -cAMP responsive element. In vascular smooth muscle cells (VSMC), activation of cAMP signaling blocks apoptosis triggered by serum deprivation. In the present study we investigated the role of gene expression in the inhibition of apoptosis by cAMP. In VSMC transfected with E1A adenovirus, incubation in the absence of serum for 6h led to 20-fold elevation of chromatin fragmentation and 10-fold activation of caspase-3 activity, these being employed as markers of apoptosis. Forskolin-induced activation of cAMP signaling was accompanied by 50% elevation of RNA synthesis and completely abol- ished the development of apoptosis during the initial 6 h incubation in growth factor-free medium. In 12 h apoptosis in forskolin-treated VSMC was slowly developed and after 24 h the content of chromatin fragments was 2-fold less than in con- trol cells. Addition of actinomycin D and cycloheximide completely blocked RNA synthesis and decreased protein synthesis by 80%, respectively. Neither compound affected baseline apoptosis or its inhibition by forskolin. More than 70 newly phos- phorylated proteins were observed by 2D-electrophoresis of VSMC after incubation with forskolin for 3 h; in 24 h the num- ber of phosphoproteins triggered by forskolin was decreased by 2-3-fold. These results show that suppression of VSMC apop- tosis under activation of cAMP signaling is mediated via posttranslational modification of pre-existing intermediates of the apoptotic machinery rather than by de novo synthesis of inhibitors of programmed cell death. Key words: apoptosis, cAMP, RNA synthesis, protein synthesis