PURINERGIC MODULATION OF ADULT GUINEA-PIG CARDIOMYOCYTES IN LONG-TERM CULTURES AND COCULTURES WITH EXTRACARDIAC OR INTRINSIC CARDIAC NEURONS

Objective: To determine the capacity of ATP to modify cardiomyocytes directly or indirectly via peripheral autonomic neurones, the effects of various purinergic agents were studied on long term cultures of adult guinea pig ventricular myocytes and their co-cultures with extracardiac (stellate ganglion) or intrinsic cardiac neurones. Methods: Ventricular myocytes and cardiac neurones were enzymatically dissociated and plated together or alone (myocytes only). Myocyte cultures were used for experiments after three to six weeks. The electrical and contractile properties of cultured myocytes and myocyte-neuronal networks were investigated. Results: The spontaneous beating frequency of ventricular myocytes co-cultured with stellate ganglion neurones increased by similar to 140% (p<0.001) following superfusion with 10(-5) M ATP. This effect was not modified significantly by tetrodotoxin or by beta adrenoceptor blockade (10(-5) M timolol), but was eliminated following application of the P-2 antagonist suramin (10(-5) M). Basal spontaneous contractile rate was reduced by similar to 86% (p<0.001) in the presence of suramin, indicating the existence of tonically active purinergic synaptic mechanisms in stellate ganglion neurone-myocyte cocultures. Suramin did not significantly affect non-innervated myocyte cultures. ATP increased myocyte contractile rate in intrinsic cardiac neurone-myocyte co-cultures by similar to 40% (p<0.01) under control conditions, but when beta adrenergic receptors of tetrodotoxin sensitive neural responses were blocked, ATP induced greater augmentation (>100%). In contrast, ATP induced much smaller effects in non-innervated myocyte cultures (similar to 26%, p<0.01). Analogues of ATP showed the following order of potency: ATP > UTP > MSATP > beta gamma ATP > alpha beta ATP. Adenosine (10(-4) M) attenuated the beating frequency of myocytes in both types of co-culture, while not significantly affecting non-innervated myocyte cultures. Conclusions: The experimental model used in this study showed that extrinsic and intrinsic cardiac neurones which possess P-2 receptors can greatly enhance cardiac myocyte contractile rate when activated by ATP. Since adenosine reduced contractile rate in both types of co-cultures while not affecting non-innervated myocytes, it is concluded that some of these neurones possess P-1 receptors.