Adreno-muscarinic Synergy in the Bladder Trigone: Calcium-dependent and 1 Independent Mechanisms. 2 3

26 27 We have recently demonstrated a strong synergy between muscarinic and 28 adrenergic excitatory pathways in the trigone which, as part of the bladder base, 29 plays a key role in outflow control. This study aimed to characterise the role of 30 intracellular Ca, [Ca]i, in this synergistic effect. Muscle strips from the superficial 31 trigone of male guinea-pigs were used for tension experiments. In isolated muscle 32 cells, [Ca]i was measured by epifluorescence microscopy using the fluorescent 33 indicator Fura-2. Phenylephrine (PE, 10 μM) augmented contractions induced by 34 carbachol (1 μM) to 4.0±0.8-fold of control, while corresponding [Ca]i levels did not 35 exceed 1.3±0.2-fold of control. Furthermore, PE generated significantly greater 36 contractions for a given rise of [Ca]i, compared to depolarising KCl solutions. The 37 protein kinase-C inhibitor GF 109203X (5 μM) and the Rho-kinase inhibitor Y-27632 38 (5 μM) reduced the PE contracture to 37.3±9.4 and 60.1±12.4 % of control, 39 respectively, without significantly altering the [Ca]i transients. GF 109203X reduced 40 the augmentation of 1 μM carbachol by PE to 1.5±0.1-fold of control. Muscarinic and 41 adrenergic receptor activation exert a powerful synergistic effect in the bladder 42 trigone without equivalent changes to the [Ca]i transient. Ca-sensitisation of 43 contractile proteins is likely to play a key role in this synergism, particularly for 44 adrenergic activation. 45 46