CHARACTERISTICS OF NITRIC OXIDE-MEDIATED CHOLINERGIC MODULATION OF CALCIUM CURRENT IN RABBIT SINOATRIAL NODE

1. We hare previously shown that nitric oxide (NO) production is essen tial for cholinergic inhibition of the beta-adrenergic stimulated L-ty pe calcium current (ICa-L) in rabbit pacemaker (sino-atrial node (SAN) ) cells. The present experiments demonstrate the presence of constitut ive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO -mediated cholinergic response. 2. Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN. 3. The a ctivation of cNOS is known to be Ca2+ and calmodulin dependent. Strong ly buffering intracellular Ca2+ with the membrane-permeable chelator B APTA-AM (10 mu M) significantly reduced (and in some cases abolished) the attenuation of ICa-L by the muscarinic agonist carbamylcholine (CC h). In contrast, the CCh-induced activation of an outward K+ current, I-K,I-ACh, was unaffected by buffering of [Ca2+](i). The calmodulin in hibitor 48/80 (20 mu M) also abolished the attenuation of ICa-L by CCh , with no change in the activation of I-K,I-ACh.4. Neither thapsigargi n nor ryanodine (5-10 mu M), agents which deplete intracellular Ca2+ s tores, significantly changed the attenuation of ICa-L by CCh. 5. Pertu ssis toxin (PTX) completely abolished both the inhibitory action of CC h on ICa-L and the activation of I-K,I-ACh. This establishes that a PT X-sensitive GTP-binding protein links the muscarinic receptor to NO sy nthase activation in SAN cells. 6. Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)-activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa-L attenuatio n. This was supported by showing that a specific inhibitor of PDE II, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), blocks the effect of CCh on ICa-L, but not on I-K,I-ACh. Using reverse transcriptase-polymeras e chain reaction techniques, we have established that PDE II is the do minant cyclic nucleotide phosphodiesterase isoform in SAN cells.