Ouabain-resistant hyperpolarization induced by insulin in aggregates of embryonic heart cells

Spheroidal aggregates formed from trypsin-dissociated 14-day embryonic chicken hearts after 48 hr of rotation on a gyratory shaker. Intracellular recorded resting membrane potentials of aggregates bathed in 1.3 mM K⁺ balanced salt solution had a mean (±SD) of 64 ± 4 mV. After a stable potential was achieved, addition of 1-100 nM sodium bovine insulin caused a slow hyperpolarization of up to 19mV after 4-5 min, followed in some cases, by a further, more rapid, shift to a potential near E(K). Equivalent hyperpolarizations were observed when insulin was added in the presence of 10 mM ouabain, indicating that enhanced Na⁺,K⁺ pump activity was not responsible for the change in membrane potential. The concentration of insulin that produced half-maximal hyperpolarization (2nM) corresponded to the association constant of a high-affinity insulin receptor, suggesting that binding to this class of receptors led to the change in membrane potential. Steady state current-voltage curves from current clamp experiments suggested that insulin produced an increase in slope conductance at potentials near rest by inducing an outward current with an apparent potential negative to -90mV.