During liver regeneration in vivo carbon monoxide (CO) and nitric oxide (NO) are supposed
to play a significant role. We raise the question whether CO and NO are involved in
the growth process of cultured hepatocytes. Rat hepatocytes were stimulated into proliferation,
growth being estimated by DNA content, mRNA by quantitative RT-PCR, and inducible
NO synthase (iNOS) activity by GC-MS. Dexamethasone proved obligatory for fast proliferation.
It suppressed the spontaneous rise of iNOS-mRNA in cultures devoid of glucocorticoids,
but did not counteract the rise in mRNA in actively dividing cultures. Expression
of iNOS-mRNA and cell growth were further enhanced by LiCl (10 mM). NOS activity was
completely suppressed by the iNOS-specific inhibitors N-(3-(aminomethyl)benzyl) acetamidine
(1400 W,100 microM) and L-N(6)-(1-iminoethyl)lysine (L-NIL, 500 microM), however,
without a decrease in hepatocyte growth. Proliferation was attenuated only by very
high concentrations (>0.5 mM) of N-nitro-L-arginine methyl ester (L-NAME) and asymmetric
dimethylarginine (ADMA). Various NO donors (at 100 microM) did not stimulate cell
growth. The furoxan CAS 1609 stimulated growth, decreased iNOS-mRNA expression and
transiently increased haem oxygenase-1 (HO-1)-mRNA without releasing considerable
amounts of NO. 1H-[1,2,4]Oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ) attenuated the
action of CAS 1609. Proliferation was stimulated by Co-protoporphyrin and tricarbonyldichlororuthenium(II)
dimer (CORM-2). We conclude that CAS 1609 triggers hepatocyte mitosis most likely
via direct, NO-independent induction of HO-1 expression, pointing to CO as a growth-promoting
signal in the proliferation cascade in cultured hepatocytes.