ROLE OF NITRIC-OXIDE IN ANTAGONISTIC EFFECTS OF TRANSFORMING GROWTH-FACTOR-BETA AND INTERLEUKIN-1-BETA ON THE BEATING RATE OF CULTURED CARDIAC MYOCYTES

We have recently shown that transforming growth factor-beta (TGFbeta) acts in an autocrine manner to maintain the beating rate of neonatal rat cardiac myocytes cultured in serum-free medium on cardiac fibroblast matrix. Interleukin-1beta (IL-1beta) suppresses the myocyte-beating rate, and TGFbeta antagonizes this effect. We now show that TGFbeta and IL-1beta also have antagonistic effects on the secretion of nitric oxide (NO) by these myocytes, and that NO secretion, the activity of NO synthase (NOS), and expression of the inducible form of NOS correlate inversely with the effects of these two agents on the beating rate. Western blot analysis shows that treatment of myocytes with TGFbeta antagonizes the induction of NOS after treatment with IL-1beta. Release of NO, induced by IL-1beta is dependent upon the availability of the substrate, L-arginine, and is suppressed by a competitive inhibitor, N(G)-monomethyl-L-arginine. L-Arginine (>0.25 mM) also suppresses, and N(G)-monomethyl-L-arginine (>0.5 mM) enhances the myocyte-beating rate. Treatment with IL-1beta, but not TGFbeta, increases cellular cGMP, presumably by activation of guanylate cyclase by NO. Methylene blue, an inhibitor of guanylate cyclase, reverses the suppression of beating caused by IL-1beta. Bacterial lipopolysaccharide, present in the serum-free medium, is a coinducer of NO secretion. The suppressive effects of NO on the beating rate can be overcome by altering either the set of cytokines employed to induce NO or the matrix on which the myocytes are cultured, demonstrating that additional parameters are also involved in regulation of the beating rate.