Primary cultures of coronary endothelial cells were obtained by enzymatic dispersion of isolated guinea-pig hearts and separation of different cardiac cell types by density gradient centrifugation. The cells were grown to confluency and the membrane potential of the monolayer was recorded using the whole-cell-clamp mode of the patch-clamp technique. 2. When superfused with physiological salt solution at 37 degrees C the average resting potential of the monolayers was -35 +/- 9 mV. A 2 min application of bradykinin (0.1-20 nM) induced a transient hyperpolarization of up to 40 mV (median 33 mV), which was followed by a sustained depolarization of up to 28 mV (median 10 mV). The average duration of the hyperpolarization, measured midway between the resting potential and peak negativity, was 48 s with 20 nM-bradykinin. 3. The concentration of bradykinin producing a half-maximal hyperpolarization was 2.5 nM. When high concentrations of bradykinin (greater than 10 nM) were applied for several minutes voltage oscillations of low amplitude with periodicity of 2-3 min were observed. 4. The peak of the hyperpolarization depended on the extracellular potassium concentration ([K+]o). The limiting slope of the relation between membrane potential and log [K+]o was 52 mV per 10-fold change in [K+]o. With 50 mM [K+]o the hyperpolarization was abolished and with 100 mM [K+]o the hyperpolarization turned into a depolarization. 5. After removal of external Ca2+ the first transient hyperpolarization elicited by bradykinin had the same amplitude as under control conditions, but its duration was reduced to about 72%. The second application of bradykinin in Ca2(+)-free solution produced only a depolarization. The hyperpolarizing response to bradykinin could be re-primed by exposing the preparation to Ca2(+)-containing solution for 2 min. 6. The transient hyperpolarization elicited by 4 nM-bradykinin could be inhibited by d-tubocurarine, a blocker of Ca2(+)-activated potassium channels. On average, 1 mM-tubocurarine reduced the hyperpolarization by 49 +/- 18%. Apamin (10 microM) reduced the hyperpolarization by 15 +/- 11%. 7. ATP (1 microM) produced a hyperpolarization of similar amplitude to that produced by bradykinin, but of shorter duration (average 29 s), and a very small (less than 5 mV) or no sustained depolarization. Histamine (10 microM) produced an even shorter transient hyperpolarization, followed by a depolarization of up to 15 mV. 8. Most of the monolayers of coronary endothelial cells responded to adenosine in a similar way as to bradykinin.(ABSTRACT TRUNCATED AT 400 WORDS)
