R56865 inhibits catecholamine release from bovine chromaffin cells by blocking calcium channels.
1. The effects of R56865 (a new class of cardioprotective agent which prevents Na+ and Ca2+ overload in cardiac myocytes) on catecholamine release, whole-cell current through Ca2+ channels (IBa) and cytosolic Ca2+ concentrations, [Ca2+]i, have been studied in bovine chromaffin cells. 2. R56865 caused a time- and concentration-dependent blockade of catecholamine release from superfused cells stimulated intermittently with 5 s pulses of 59 mM K+. After 5 min superfusion, a 3 microM concentration inhibited secretion by 20%; the blockade increased gradually with perfusion time, to reach 85% after 40 min. The IC50 to block secretion after 5 min periods of exposure to increasing concentrations of R56865 was around 3.1 microM. The blocking effects of R56865 were reversible after 5-15 min wash out. In high Ca2+ solution (10 mM Ca2+), the degree of blockade of secretion diminished by 20% in comparison with 1 mM Ca2+. 3. In electroporated cells, R56865 (10 microM) did not modify the secretory response induced by the application of 10 microM free Ca2+. 4. R56865 blocked the peak IBa current in a concentration- and time-dependent manner; its IC50 was very similar to that obtained for secretion (3 microM). The compound not only reduced the size of the peak current but also promoted its inactivation; when the effects of R56865 were measured at the most inactivated part of the current, its IC50 was 1 microM. Both the inactivation and the reduction of the peak currents were reversible upon washing out the drug. 5. In fura-2-loaded single chromaffin cells the basal [Ca2+]i of around 100 nM was elevated to a peak of1.5 microM by the application of a 5 s pulse of 59 mM K+. R56865 (10 microM) did not affect the basal [Ca2+]but blocked by 90% the K+-evoked increase. This effect was fully reversible after 5-10 min of wash out.6. The results are compatible with the idea that R56865 blocks Ca2+ entry into K+-depolarized chromaffin cells by promoting the inactivation of voltage-dependent Ca2+ channels; this would lead to the limitation of the rise in [Ca2+]i and of the release of catecholamines. The restriction of catecholamine release may favour indirectly the known direct beneficial cardioprotective actions of R56865.
Garcez-Do-Carmo
L
L
Departmento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
Albillos
A
A
Artalejo
A R
AR
de la Fuente
M T
MT
López
M G
MG
Gandía
L
L
Michelena
P
P
García
A G
AG
D000818:Animals; D052160:Benzothiazoles; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002838:Chromaffin System / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D018274:Electroporation; D006706:Homeostasis / Q000187:drug effects; D010880:Piperidines / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology*
D052160:Benzothiazoles; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002395:Catecholamines; D010880:Piperidines; D013844:Thiazoles; C054551:R 56865; D011188:Potassium; D002118:Calcium