BETA-ADRENERGIC AND CHOLINERGIC MODULATION OF THE INWARDLY RECTIFYINGK-PIG VENTRICULAR MYOCYTES( CURRENT IN GUINEA)

1. Whole-cell patch-clamp technique was used to study the beta-adrener gic and cholinergic regulation of the inwardly rectifying K+ conductan ce (g(K1)) in isolated guinea-pig ventricular myocytes. 2. In Cl--free solutions or in the presence of 9-anthracenecarboxylic acid or Co2+, bath-applied isoprenaline (Iso) partially inhibited the steady-state w hole-cell conductance (g(ss)) calculated from the steady-state current (I-ss)-voltage (I-ss - V) curve at membrane voltages (V-m) negative t o the equilibrium potential for potassium (E(K)). I-ss was also inhibi ted at V-m positive to E(K) when the extracellular [K+] was 20 mM. The Iso-sensitive component of g(ss) exhibited the characteristics of the inwardly rectifying K+ conductance (g(K1)). 3. The Iso-induced inhibi tion of g(K1) was reversible, concentration dependent, blocked by prop ranolol, mimicked by both forskolin and dibutyryl cAMP, and prevented by including a cAMP-dependent protein kinase (PKA) inhibitor in the pi pette solution. These findings suggest that PKA mediates the Iso-induc ed inhibition of g(K1). 4. The apparent dissociation constant (K-D) fo r the concentration dependence of Iso-induced inhibition was 0.035 mu M and the Hill coefficient was similar to 1.0. A maximal Iso concentra tion (1 mu M) inhibited g(K1) by 40 +/- 4.1% (mean +/- S.E.M.; n = 13) . 5. Bath application of acetylcholine (ACh, 0.1 mu M or more) antagon ized the Iso-induced (1 mu M) inhibition of g(K1); [ACh] > 1.0 mu M an tagonized 88 +/- 2.1 % (n = 10) of the inhibition. ACh increased the K -D for Iso to inhibit Iso-sensitive g(K1) and also reduced the maximal Iso-induced inhibition. 6. ACh-induced antagonism could be abolished by pre-incubating myocytes with -pertussis toxin (PTX), suggesting tha t a muscarinic receptor-coupled, PTX-sensitive G protein, G(i), is inv olved. 7. ACh (10 mu M) also antagonized similar to 70% of the dibutyr yl cyclic AMP (1 mM)-induced inhibition of g(K1) (n = 3), suggesting t hat the ACh-induced antagonism involves more than simply inhibiting th e Iso-mediated activation of adenylyl cyclase via the activated G(i). 8. Intracellularly applied okadaic acid (OkA, 1 mu M) did not alter g( K1) (control = 134 +/- 5.1 nS vs. OkA = 136 +/- 6.1 nS), but the Iso-i nduced decrease in g(K1) was less (P < 0.001) with OkA present (42.1 /- 2.4 nS, n = 5) than when absent (54.0 +/- 2.2 nS, n = 10). However, ACh (10 mu M) failed to antagonize Iso-induced inhibition with OkA pr esent, suggesting involvement of a protein phosphatase.