B₂ Bradykinin Receptors in Cultured Neonatal Rat Cardiomyocytes Mediate a Negative Chronotropic and Negative Inotropic Response

Receptors for bradykinin (BK) were characterized in primary cultures of beating neonatal rat cardiomyocytes using [³H]BK as radioligand. Degradation studies demonstrated that [³H]BK was stable for at least 2 h when incubated with cardiomyocytes at 2 and 37°C in the presence of bacitracin in combination with captopril or ramiprilat. Without these inhibitors, >80% of the [³H]BK was degraded within 2 h at 37°C. This indicates that angiotensin-converting enzyme (ACE) is responsible for the main BK-degrading activity in cardiomyocytes. Scat-chard plots were linear and gave a K_d of 1.5 ± 0.8 nmol/1 (mean ± SD, n = 4) and a maximum binding capacity of 55–125 fmol/mg protein. Association and dissociation studies showed that binding of [³H]BK was saturable and reversible. Binding of [³H]BK at 37°C led to internaliza-tion of the ligand. Competition studies with B₁ and B₂agonists and antagonists were consistent with a B₂ subtype of receptor. Addition of BK to beating cardiomyocytes (>1 nmol/1) at 37°C gave a strong but transient negative chronotropic effect. This response was paralleled by changes in the pulsation amplitude, which indicated a simultaneous negative inotropic effect of BK. These results provide a basis for the hypothesis that ACE inhibition exerts its cardioprotective effect at the level of a population of cardiomyocytes by virtue of kinin receptor-mediated mechanisms.